Biological devices for producing oxidized zinc and applications thereof

ABSTRACT

Described herein are biological devices and methods for using the same to produce oxidized zinc. The biological devices include microbial cells transformed with a DNA construct containing genes for producing a zinc-related protein, an alkaline phosphatase, and an alcohol dehydrogenase. In some instances, the biological devices also include a gene for lipase. The oxidized zinc compositions produced herein have numerous applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority upon U.S. provisional applicationSerial Nos. 62/557,340 filed on Sep. 12, 2017 and 62/650,356 filed Mar.30, 2018. These applications are hereby incorporated by reference intheir entirety.

CROSS REFERENCE TO SEQUENCE LISTING

The genetic components described herein are referred to by sequenceidentifier numbers (SEQ ID NO). The SEQ ID NOs correspond numerically tothe sequence identifiers <400>1, <400>2, etc. The Sequence Listing, inwritten computer readable format (CRF), is incorporated by reference inits entirety.

BACKGROUND

Zinc oxide is widely used across a variety of industries. Itsultraviolet-absorbing properties have been exploited in materials suchas paints and coatings, plastics, and even sunscreen creams. Zinc oxideis employed industrially in the curing and vulcanization of rubber andlatex, to impart heat resistance and abrasion resistance to rubber andplastic products, and in the medical industry in applications from woundhealing to dental cement. Further, zinc oxide is a precursor materialfor other zinc salts including zinc diacrylate (used in the manufactureof golf balls), zinc chromate (used for anti-corrosion purposes), zincborate and zinc chloride (used as flame retardants), zinc gluconate(used in cold-prevention lozenges and sprays), and zinc dithiophosphate(an anti-wear ingredient in lubricants), among others.

Production of zinc oxide can be expensive. Zinc ores must first beground and sometimes roasted to produce zinc oxide. Sulfur dioxide is atypical byproduct of the process of roasting zinc ores; when thiscompound is released into the atmosphere, it contributes to acid rain.Toxic cadmium vapor is another byproduct of zinc refining, and further,zinc mining operations can lead to significant levels of heavy metalpollution in the air, in soil, and in waterways, causing concerns forhuman health, agriculture, and wildlife. Furthermore, the use of impuresources of zinc can lead to impurities and discoloration in the finalproduct, affecting its use as a pigment, and can cause material toaggregate in processing furnaces and other equipment, reducing the lifetime of said equipment. Finally, although zinc oxide is non-toxic, fumessuch as those generated when zinc and/or zinc alloys are vaporized ormelted and oxidized can be quite hazardous, as well.

What is needed is a new method for producing oxidized zinc. Ideally, themethod would be inexpensive to conduct and would work with a variety ofstarting materials including recycled materials and impure zinc sources.Furthermore, the method would not require the use of harsh chemicals orhigh-temperature furnaces and would generate fewer health andenvironmental hazards than traditional methods for producing zinc oxide.

SUMMARY

Described herein are biological devices and methods for using the sameto produce oxidized zinc. The biological devices include microbial cellstransformed with a DNA construct containing genes for producing azinc-related protein, an alkaline phosphatase, and an alcoholdehydrogenase. In some instances, the biological devices also include agene for lipase. The oxidized zinc compositions produced herein havenumerous applications.

The advantages of the invention will be set forth in part in thedescription that follows, and in part will be obvious from thedescription, or may be learned by practice of the aspects describedbelow. The advantages described below will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIGS. 1A and 1B show, respectively, a linear and circular schematic of aconstructed pYES2 plasmid showing the direction, placement, and size ofgenetic parts used of an exemplary DNA device described herein.

FIGS. 2A and 2B show, respectively, a linear and circular schematic of aconstructed pYES2 plasmid showing the direction, placement, and size ofgenetic parts used of an exemplary DNA device described herein.

FIGS. 3A and 3B show two replicates of a Bacillus subtilis culture after24 hours and before exposure to UV radiation.

FIGS. 4A-C show bacterial growth after UV exposure. FIG. 4A represents30 minutes of UV exposure, FIG. 4B represents 1 hour of UV exposure, andFIG. 4C represents 24 hours of UV exposure. In each, the leftmost petridish is a control bacterial culture without treatment, the center petridish is a low dilution of a bacterial culture treated with the extractsdisclosed herein, and the right petri dish is a high dilution of abacterial culture treated with the extracts disclosed herein.

FIGS. 5A-D show Bacillus subtilis cultures before and after exposure toUV light. FIG. 5A represents a culture prior to UV exposure. FIG. 5Brepresents bacterial cultures after 30 minutes of UV exposure in anuntreated control (left panel) and a culture treated with the extractsdisclosed herein (right panel). FIG. 5C represents bacterial culturesafter 1 hour of UV exposure in an untreated control (left panel) and aculture treated with the extracts disclosed herein (right panel). FIG.5D represents the same cultures, respectively, after 24 hours ofexposure to UV light.

FIGS. 6A-B show B. subtilits cultures before and after exposure to UVlight. FIG. 6A represents a culture prior to UV exposure treated with<100 nm zinc oxide nanopowder. FIG. 6B represents this same cultureafter 30 minutes of UV exposure (left panel), 1 hour of UV exposure(center panel), and after 24 hours of UV exposure (right panel).

FIGS. 7A-D show the process and results of an electrochemical analysisof zinc oxide nanopowder. FIG. 7A shows zinc oxide nanopowder (<100 nmparticle size), which dissolves at pH 2. FIG. 7B shows an extract fromthe devices disclosed herein, which dissolves at a pH of 6.89. FIG. 7Cshows an acid digestion of zinc oxide nanopowder (required fordissolution of the sample). FIG. 7D shows an acid digestion of extractsfrom the devices disclosed herein.

FIGS. 8A-B show output from a voltamperimeter for various experimentalsamples and controls. FIG. 8A represents current (in A) versus potentialdifference (in V) for zinc nanopowder in nitric acid. FIG. 8B representscurrent versus potential difference for the undigested extractsdisclosed herein.

FIGS. 9A-F show calibration curves (current in A versus concentration ing/L) useful in the determination of zinc concentration in the devicesdisclosed herein. FIG. 9A is a standard curve for zinc. FIG. 9B is astandard curve for zinc oxide in 65% nitric oxide. FIG. 9C is a standardcurve for the zinc oxide nanopowder used as a control herein. FIG. 9D isa standard curve for extracts produced by the biological devicesdisclosed herein. FIG. 9E is a standard curve for the extracts producedfrom the biological devices disclosed herein for lower concentrationsthan in FIG. 9D, wherein the samples were not filtered. FIG. 9F is astandard curve for the extracts produced by the biological devicesdisclosed herein wherein the samples were filtered with a 0.2 μm nylonfilter prior to analysis.

DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theaspects described below are not limited to specific compounds, syntheticmethods, or uses, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to be limiting.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a restriction enzyme” includes mixtures of two or moresuch restriction enzymes, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally includes a reporterprotein” means that the reporter protein may or may not be present.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

Disclosed are materials and components that can be used for, can be usedin conjunction with, can be used in preparation for, or are products ofthe disclosed compositions and methods. These and other materials aredisclosed herein, and it is understood that when combinations, subsets,interactions, groups, etc., of these materials are disclosed, that whilespecific reference to each various individual and collective combinationand permutation of these compounds may not be explicitly disclosed, eachis specifically contemplated and described herein. For example, if abacterium is disclosed and discussed and a number of differentcompatible bacterial plasmids are discussed, each and every combinationand permutation of bacterium and bacterial plasmid that is possible isspecifically contemplated unless specifically indicated to the contrary.For example, if a class of molecules A, B, and C are disclosed as wellas a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited, each is individually and collectively contemplated. Thus, inthis example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D,C-E, and C-F are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. Likewise, any subset or combination of these is alsospecifically contemplated and disclosed. Thus, for example, thesub-group of A-E, B-F, and C-E is specifically contemplated and shouldbe considered disclosed from disclosure of A, B, and C; D, E, and F; andthe example combination A-D. This concept applies to all aspects of thisdisclosure including, but not limited to, steps in methods of making andusing the disclosed compositions. Thus, if there are a variety ofadditional steps that can be performed, it is understood that each ofthese additional steps can be performed with any specific embodiment orcombination of embodiments of the disclosed methods, and that each suchcombination is specifically contemplated and should be considereddisclosed.

Described herein is a process for producing oxidized zinc usingmicrobial cells that includes (a) making a DNA construct containinggenes for producing a zinc-related protein, an alkaline phosphatase, andan alcohol dehydrogenase, (b) introducing the DNA construct into hostmicrobial cells via transformation or transfection, and (c) culturingthe microbial host cells to produce oxidized zinc.

I. DNA Constructs

DNA constructs are provided herein for the production of oxidized zinc.It is understood that one way to define the variants and derivatives ofthe genetic components and DNA constructs described herein is in termsof homology/identity to specific known sequences. Those of skill in theart readily understand how to determine the homology of two nucleicacids. For example, the homology can be calculated after aligning thetwo sequences so that the homology is at its highest level. Another wayof calculating homology can be performed according to publishedalgorithms (see Zuker, M., Science, 244:48-52, 1989; Jaeger et al.,Proc. Natl. Acad. Sci. USA, 86:7706-7710, 1989; and Jaeger et al.,Methods Enzymol., 183:281-306, 1989, which are herein incorporated byreference for at least material related to nucleic acid alignment.

As used herein, “conservative” mutations are mutations that result in anamino acid change in the protein produced from a sequence of DNA. When aconservative mutation occurs, the new amino acid has similar propertiesas the wild type amino acid and generally does not drastically changethe function or folding of the protein (e.g., switching isoleucine forvaline is a conservative mutation since both are small, branched,hydrophobic amino acids). “Silent mutations,” meanwhile, change thenucleic acid sequence of a gene encoding a protein but do not change theamino acid sequence of the protein.

It is understood that the description of mutations and homology can becombined together in any combination, such as embodiments that have atleast 70%, 75%, 80%, 85%, 90%, 95%, or 99% homology to a particularsequence wherein the variants are conservative or silent mutations. Itis understood that any of the sequences described herein can be avariant or derivative having the homology values listed above.

In one aspect, a database such as, for example, GenBank, can be used todetermine the sequences of genes and/or regulatory regions of interest,the species from which these elements originate, and related homologoussequences.

In one aspect, genes of interest can be incorporated into a DNAconstruct. In a further aspect, the DNA construct can be incorporated aspart of a vector for transfection into microbial cells. In a stillfurther aspect, the vector can be a plasmid, a phagemid, a cosmid, ayeast artificial chromosome, a bacterial artificial chromosome, a virus,a phage, or a transposon. In another aspect, the microorganisms arefungi or bacteria. In one aspect, the fungi are yeasts such as, forexample, Saccharomyces cerevisiae. In another aspect, the bacteria areEscherichia coli.

Vectors capable of high levels of expression of recombinant genes andproteins are well known in the art. Vectors useful for thetransformation of a variety of host cells are common and commerciallyavailable and include, for example, pWLneo, pSV2cat, pOG44, pXT1, pSG,pSVK3, pBSK, pBR322, pYES, pYES2, pBSKII, and pUC. The skilledpractitioner will be able to choose a plasmid based on such factors as(a) the amount of nucleic acid (i.e., number of genes and otherelements) to be inserted, (b) the host organism, (c) culture conditionsfor the host organism, and other related factors.

In one aspect, the DNA construct includes the following geneticcomponents: (a) a gene that expresses zinc-related protein; (b) a genethat expresses alkaline phosphatase; and (c) a gene that expressesalcohol dehydrogenase.

In one aspect, the nucleic acids (e.g., genes that express zinc-relatedprotein, alkaline phosphatase, and alcohol dehydrogenase) used in theDNA constructs described herein can be amplified using polymerase chainreaction (PCR) prior to being ligated into a plasmid or other vector.Typically, PCR amplification techniques make use of primers, or short,chemically-synthesized oligonucleotides that are complementary toregions on each respective strand flanking the DNA or nucleotidesequence to be amplified. A person having ordinary skill in the art willbe able to design or choose primers based on the desired experimentalconditions. In general, primers should be designed to provide for bothefficient and faithful replication of the target nucleic acids. Twoprimers are required for the amplification of each gene, one for thesense strand (that is, the strand containing the gene of interest) andone for the antisense strand (that is, the strand complementary to thegene of interest). Pairs of primers should have similar meltingtemperatures that are close to the PCR reaction's annealing temperature.In order to facilitate the PCR reaction, the following features shouldbe avoided in primers: mononucleotide repeats, complementarity withother primers in the mixture, self-complementarity, and internalhairpins and/or loops. Methods of primer design are known in the art;additionally, computer programs exist that can assist the skilledpractitioner with primer design. Primers can optionally incorporaterestriction enzyme recognition sites at their 5′ ends to assist in laterligation into plasmids or other vectors.

PCR can be carried out using purified DNA, unpurified DNA that isintegrated into a vector, or unpurified genomic DNA. The process foramplifying target DNA using PCR consists of introducing an excess of twoprimers having the characteristics described above to a mixturecontaining the sequence to be amplified, followed by a series of thermalcycles in the presence of a heat-tolerant or thermophilic DNApolymerase, such as, for example, any of Taq, Pfu, Pwo, Tfl, rTth, Tli,or Tma polymerases. A PCR “cycle” involves denaturation of the DNAthrough heating, followed by annealing of the primers to the target DNA,followed by extension of the primers using the thermophilic DNApolymerase and a supply of deoxynucleotide triphosphates (i.e., dCTP,dATP, dGTP, and TTP), along with buffers, salts, and other reagents asneeded. In one aspect, the DNA segments created by primer extensionduring the PCR process can serve as templates for additional PCR cycles.Many PCR cycles can be performed to generate a large concentration oftarget DNA or gene. PCR can optionally be performed in a device ormachine with programmable temperature cycles for denaturation,annealing, and extension steps. Further, PCR can be performed onmultiple genes simultaneously in the same reaction vessel ormicrocentrifuge tube since the primers chosen will be specific toselected genes. PCR products can be purified by techniques known in theart such as, for example, gel electrophoresis followed by extractionfrom the gel using commercial kits and reagents.

In a further aspect, the plasmid can include an origin of replication,allowing it to use the host cell's replication machinery to createcopies of itself.

As used herein, “operably linked” refers to the association of nucleicacid sequences on a single nucleic acid fragment so that the function ofone affects the function of another. For example, if sequences formultiple genes are inserted into a single plasmid, their expression maybe operably linked. Alternatively, a promoter is said to be operablylinked with a coding sequence when it is capable of affecting theexpression of that coding sequence.

As used herein, “expression” refers to transcription and/or accumulationof an mRNA derived from a gene or DNA fragment. Expression may also beused to refer to translation of mRNA into a peptide, polypeptide, orprotein.

In one aspect, the gene that expresses zinc-related protein is isolatedfrom an animal. In a further aspect, the animal is a fish such as, forexample, Atlantic salmon. In an alternative aspect, the gene thatexpresses zinc-related protein is isolated from a bacterium. In oneaspect, the bacterium is a Streptomyces, Polaribacter, Kitasatospora,Actinobacter, Azospirillum, Clostridium, or Collimonas, Micromonosporaspecies. In a still further aspect, the gene that expresses zinc-relatedprotein is isolated from an alga. In one aspect, the alga is Guillardiatheta. In a further aspect, the gene that expresses zinc-related proteinhas SEQ ID NO. 1 or at least 70% homology thereto, at least 75% homologythereto, at least 80% homology thereto, at least 85% homology thereto,at least 90% homology thereto, or at least 95% homology thereto. Inanother aspect, the zinc-related protein is calmodulin or anotherzinc-binding protein, or a homolog thereof. In one aspect, the gene thatexpresses zinc-related protein is isolated from Streptomyceszinciresistens and can be found in GenBank with GI number EGX59011.1.

Other sequences expressing zinc-related protein or related or homologousgenes can be identified in a database such as, for example, GenBank. Inone aspect, sequences useful herein include those with the GI numberslisted in Table 1:

TABLE 1 Zinc-Related Protein Genes Sequence Source Organism DescriptionGI Number Streptomyces lincolnensis genomic DNA CP016438.1 Streptomycessp. 4F genomic DNA CP013142.1 Streptomyces collinus genomic DNACP006259.1 Streptomyces avermitilis genomic DNA BA000030.4 Streptomycessp. 3124.6 genomic DNA LT670819.1 Streptomyces sp. 1H-SSA4 genomic DNACP022161.1 Streptomyces parvulus genomic DNA CP015866.1 Streptomycesambofaciens genomic DNA CP012949.1 Streptomyces ambofaciens genomic DNACP012382.1 Streptomyces scabiei genomic DNA FN554889.1 Streptomycesdavawensis genomic DNA HE971709.1 Polaribacter sp. SA4-12 genomic DNACP019334.1 Streptomyces sp. 11-1-2 genomic DNA CP022545.1 Streptomycessp. CdTB01 genomic DNA CP013743.1 Kitasatospora setae genomic DNAAP010968.1 Streptomyces pluripotens genomic DNA CP022433.1 Streptomycespluripotens genomic DNA CP021080.1 Streptomyces pactum genomic DNACP019724.1 Polaribacter sp. Hell_33_78 genomic DNA LT629794.1Streptomyces sp. TLI_053 genomic DNA LT629775.1 Streptomyces pactumgenomic DNA CP016795.1 Streptomyces puniciscabiei genomic DNA CP017248.1Streptomyces griseochromogenes genomic DNA CP016279.1 Streptomycesincarnates genomic DNA CP011497.1 Kitasatospora auregfaciens genomic DNACP020567.1 Streptomyces sp. S10(2016) genomic DNA CP015098.1Streptomyces reticuli genomic DNA LN997842.1 Actinobacteria bacteriumIMCC25003 genomic DNA CP015603.1 Polaribacter sp. KT25b genomic DNALT629752.1 Streptomyces hygroscopicus genomic DNA CP013219.1Streptomyces sp. Mg1 genomic DNA CP011664.1 Azospirillum brasilensegenomic DNA CP007796.1 Streptomyces hygroscopicus genomic DNA CP003720.1Streptomyces hygroscopicus genomic DNA CP003275.1 Clostridum cochleariumgenomic DNA LT906477.1 Collimonas arenae genomic DNA CP009962.1Polaribacter sp. MED152 genomic DNA CP004349.1 Streptomyces sp. S8genomic DNA CP015362.1 Micromonospora echinofusca genomic DNA LT607733.1Streptomyces sp. PBH53 genomic DNA CP011799.1 Streptomyces fulvissimusgenomic DNA CP005080.1 Streptomyces katrae genomic DNA CP020042.1Streptomyces silaceus genomic DNA CP015588.1 Streptomyces venezuelaegenomic DNA CP018074.1 Salmo solar calmodulin XM_014213459.1Streptomyces venezuelae genomic DNA FR845719.1 Salmo solar calmodulinBT059493.1 Salmo solar calmodulin BT045544.1 Streptomyces albireticuligenomic DNA CP021744.1 Streptomyces sp. 3211 genomic DNA CP020039.1Clostridium sporogenes genomic DNA CP011663.1 Clostridium sporogenesgenomic DNA CP009225.1 Clostridium botulinum genomic DNA CP006902.1Guillardia theta genomic DNA XM_005830304.1

In one aspect, the gene that expresses alkaline phosphatase is isolatedfrom an insect. In a further aspect, the insect is a fruit fly such as,for example, from the genus Ceratitis. In another aspect, the gene thatexpresses alkaline phosphatase is isolated from a fungus. In a furtheraspect, the fungus can be a pathogenic or non-pathogenic fungus, afungus that forms a symbiotic relationship with plant roots, a yeast, aslime mold, or a mitosporic fungus. In a still further aspect, thefungus can be from the genus Vanderwaltozyma, Dactylellina,Funneliformis, Gigaspora, Cyberlindnera, Schizosaccharomyces, Candida,Polysphondylium, Trichophyton, Lobosporangium, Hyphopichia, or anothercommon fungal genus. In a still another aspect, the gene that expressesalkaline phosphatase is isolated from a bacterium. In one aspect, thebacterium can be Gram-negative or Gram-positive, can be aerobic oranaerobic, can be spore-forming or non-spore forming, can be motile orsessile, can have a bacillus or coccus shape, can be found in afreshwater or marine environment or associated with the microbiome of ahuman or other animal, or can be adapted to an extreme condition such ascold or a hyperthermophilic environment. In one aspect, the bacterium isfrom one of the following genera: Haliscomenobacter, Alkalitalea,Owenweeksia, Porphyromonadaceae, Aequorivita, Alteromoas, Polaribacter,Enterococcus, Xenorhabdus, Bacteroides, Maribacter, Thermotoga,Dokdonia, Bacillus, Sphingobacterium, Mucilaginibacter, Cellulophaga, orGlaciecola. In a further aspect, the gene that expresses alkalinephosphatase has SEQ ID NO. 2 or at least 70% homology thereto, at least75% homology thereto, at least 80% homology thereto, at least 85%homology thereto, at least 90% homology thereto, or at least 95%homology thereto. In one aspect, the gene that expresses an alkalinephosphatase is isolated from Haliscomenobacter hydrossis and can befound in GenBank with GI number AEE52072.1.

Other sequences expressing alkaline phosphatase or related or homologousgenes can be identified in a database such as, for example, GenBank. Inone aspect, sequences useful herein include those with the GI numberslisted in Table 2:

TABLE 2 Alkaline Phosphatase Genes Source Organism Sequence DescriptionGI Number Haliscomenobacter genomic DNA CP002691.1 hydrossis Alkalitaleasaponilacus genomic DNA CP021904.1 Uncultured bacterium genomic DNAKU516288.1 Uncultured bacterium genomic DNA KU516232.1 Unculturedbacterium genomic DNA KU516199.1 Uncultured bacterium genomic DNAKU516115.1 Owenweeksia genomic DNA CP003156.1 hongkongensisVanderwaltozyma hypothetical protein XM_001645979.1 polysporaDactylellina haptotyla hypothetical protein XM_011111730.1Porphyromonadaceae genomic DNA LN515532.1 bacterium Aequorivitasublithincola genomic DNA CP003280.1 Alteromonas macleodii genomic DNACP014323.1 Alteromonas macleodii genomic DNA CP003873.1 Polaribacter sp.genomic DNA CP019334.1 Funneliformis mosseae alkaline phosphataseCP002528.1 Enterococcus faecalis genomic DNA JX997747.1 Enterococcusfaecalis genomic DNA CP022712.1 Enterococcus faecalis genomic DNACP015883.1 Enterococcus faecalis genomic DNA CP021161.1 Enterococcusfaecalis genomic DNA CP015410.2 Enterococcus faecalis genomic DNACP019512.1 Enterococcus faecalis genomic DNA CP015998.1 Enterococcusfaecalis genomic DNA CP018102.1 Enterococcus faecalis genomic DNAAP017623.1 Enterococcus faecalis genomic DNA CP014949.1 Enterococcusfaecalis genomic DNA CP008816.1 Xenorhabdus poinarii genomic DNAFO704551.1 Enterococcus faecalis genomic DNA CP004081.1 Enterococcusfaecalis genomic DNA HF558530.1 Enterococcus faecalis genomic DNACP003726.1 Enterococcus faecalis genomic DNA CP002621.1 Enterococcusfaecalis genomic DNA CP002491.1 Enterococcus sp. genomic DNA FP929058.1TnphoZ mutagenesis vector genomic DNA AY028776.1 Enterococcus faecalisgenomic DNA AE016830.1 Cloning vector genomic DNA AF167172.1 Bacteroideshelcogenes genomic DNA CP002352.1 Maribacter sp. genomic DNA CP002157.1Gigaspora margarita alkaline phosphatase AB114299.1 Cyberlindnerafabianii genomic DNA LK052911.1 Thermotoga naphthophila genomic DNACP001839.1 Thermotoga sp. genomic DNA CP000969.1 Thermotoga sp. 16S RNAAJ872273.1 Thermotoga naphthophila 16S RNA AJ872268.1 Enterococcusfaecalis genomic DNA CP018004.1 Dokdonia donghaensis genomic DNACP015125.1 Bacillus simplex genomic DNA CP011008.1 Schizosaccharomycesvacuolar membrane NM_001022665.2 pombe alkaline phosphatase Candidadubliniensis alkaline phosphatase XM_002417429.1 precursor Candidadubliniensis genomic DNA FM992688.1 Schizosaccharomyces genomic DNACU329671.1 pombe Schizosaccharomyces alkaline phosphatase AF316541.1pombe Polysphondylium pallidum alkaline phosphatase XM_020573858.1Bacillus anthracis genomic DNA CP019726.1 Proteiniphilum genomic DNALT605205.1 saccharofermetans Bacillus cereus genomic DNA CP018935.1Bacillus cereus genomic DNA CP018931.1 Bacillus anthracis genomic DNAAP014833.1 Bacillus cereus genomic DNA CP009605.1 Bacillus thuringiensisgenomic DNA CP010088.1 Bacillus anthracis genomic DNA CP009981.1Bacillus cereus genomic DNA CP009968.1 Bacillus anthracis genomic DNACP009902.1 Bacillus thuringiensis genomic DNA CP009720.1 Bacillusanthracis genomic DNA CP009598.1 Bacillus cereus genomic DNA CP009596.1Bacillus anthracis genomic DNA CP009700.1 Bacillus anthracis genomic DNACP009544.1 Bacillus anthracis genomic DNA CP009541.1 Bacillus anthracisgenomic DNA CP009476.1 Bacillus anthracis genomic DNA CP009464.1Bacillus anthracis genomic DNA CP009331.1 Bacillus anthracis genomic DNACP009325.1 Bacillus anthracis genomic DNA CP008752.1 Bacillus anthracisgenomic DNA CP007618.1 Bacillus cereus genomic DNA CP003747.1 Bacilluscoagulans genomic DNA CP003056.1 Trichophyton verrucosum hypotheticalprotein XM_003019232.1 Tricophyton benhamiae hypothetical proteinXM_003016737.1 Bacillus anthracis genomic DNA EF039850.1Sphingobacterium mizutaii genomic DNA LT906468.1 Sphingobacteriaceaegenomic DNA CP021237.1 bacterium Ceratitis capitate membrane-boundXM_004522406.3 alkaline phosphatase Candida tanzawaensis alkalinephosphatase- XM_020211106.1 like protein Mucilaginibacter sp. genomicDNA CP014773.1 Thermotoga maritima genomic DNA CP011108.1 Thermotogamaritima genomic DNA CP011107.1 Thermotoga maritima genomic DNACP010967.1 Cellulophaga baltica genomic DNA CP009976.1 Thermotoga sp.genomic DNA CP003409.1 Dokdonia sp. genomic DNA CP009301.1 Thermotogamaritima genomic DNA CP007013.1 Thermotoga maritima genomic DNACP004077.1 Glaciecola psychrophila genomic DNA CP003837.1 Cellulophagaalgicola genomic DNA CP002453.1 Thermotoga petrophila genomic DNACP000702.1 Thermotoga maritima genomic DNA AE000512.1 Lobosporangiumalkaline phosphatase- XM_022029226.1 transversal like protein Bacillushorikoshii genomic DNA CP020880.1 Hyphopichia burtonii alkalinephosphatase- XM_020222701.1 like protein

In one aspect, the gene that expresses alcohol dehydrogenase is isolatedfrom a fungus. In a further aspect, the fungus is a yeast such as, forexample, Saccharomyces cerevisiae. In a still further aspect, the S.cerevisiae is from strain S288c, N85, Y12, ySR127, AHY0914, YJM451,YJM470, YJM554, YJM555, YJM682, YJM689, YJM972, YJM975, YJM978, YJM996,YJM1083, YJM1133, YJM1190, YJM1208, YJM1250, YJM1307, YJM1356, YJM1381,YJM1383, YJM1385, YJM1386, YJM1388, YJM1389, YJM1419, YJM1433, YJM1456,YJM1460, YJM1526, YJM1592, or YJM1615. In an alternative aspect, the S.cerevisiae is a wild type strain. In a further aspect, the gene thatexpresses alcohol dehydrogenase has SEQ ID NO. 3 or at least 70%homology thereto, at least 75% homology thereto, at least 80% homologythereto, at least 85% homology thereto, at least 90% homology thereto,or at least 95% homology thereto. In one aspect, the gene that expressesalcohol dehydrogenase is isolated from Saccharomyces cerevisiae and canbe found in GenBank with GI number J01314.1.

Other sequences expressing alcohol dehydrogenase or related orhomologous genes can be identified in a database such as, for example,GenBank. In one aspect, sequences useful herein include those with theGI numbers listed in Table 3:

TABLE 3 Alcohol Dehydrogenase Genes Source Organism Sequence DescriptionGI Number Saccharomyces cerevisiae alcohol dehydrogenase II J01314.1Saccharomyces cerevisiae chromosome XIII sequence CP005453.2Saccharomyces cerevisiae chromosome XIII sequence CP020135.1Saccharomyces cerevisiae chromosome XIII sequence CP005452.2Saccharomyces cerevisiae chromosome XIII sequence CP005450.2Saccharomyces cerevisiae chromosome XIII sequence BK006946.2Saccharomyces cerevisiae chromosome XIII sequence Z49212.1 Saccharomycescerevisiae alcohol dehydrogenase II NM_001182812.1 Saccharomycescerevisiae alcohol dehydrogenase II EF059086.1 Saccharomyces cerevisiaealcohol dehydrogenase M38457.1 Saccharomyces cerevisiae chromosome XIIIsequence CP005464.2 Saccharomyces cerevisiae chromosome XIII sequenceCP005483.2 Saccharomyces cerevisiae chromosome XIII sequence CP005432.2Saccharomyces cerevisiae chromosome XIII sequence CP005482.2Saccharomyces cerevisiae chromosome XIII sequence LN907796.1Saccharomyces cerevisiae chromosome XIII sequence CP005456.1Saccharomyces cerevisiae chromosome XIII sequence CP005455.1Saccharomyces cerevisiae chromosome XIII sequence CP005440.1Saccharomyces cerevisiae chromosome XIII sequence CP020203.1Saccharomyces cerevisiae chromosome XIII sequence CP005403.2Saccharomyces cerevisiae chromosome XIII sequence CP005472.2Saccharomyces cerevisiae chromosome XIII sequence CP005465.2Saccharomyces cerevisiae chromosome XIII sequence CP005405.2Saccharomyces cerevisiae chromosome XIII sequence CP005414.2Saccharomyces cerevisiae chromosome XIII sequence CP005412.2Saccharomyces cerevisiae chromosome XIII sequence CP005451.2Saccharomyces cerevisiae chromosome XIII sequence CP011559.1Saccharomyces cerevisiae chromosome XIII sequence CP005436.1Saccharomyces cerevisiae chromosome XIII sequence CP005426.1Saccharomyces cerevisiae chromosome XIII sequence CP005406.1Saccharomyces cerevisiae alcohol dehydrogenase II JX901290.1Saccharomyces cerevisiae chromosome XIII sequence CP020169.1Saccharomyces cerevisiae chromosome XIII sequence CP005449.2Saccharomyces cerevisiae chromosome XIII sequence CP005429.2Saccharomyces cerevisiae chromosome XIII sequence CP005419.2Saccharomyces cerevisiae chromosome XIII sequence CP005409.2Saccharomyces cerevisiae chromosome XIII sequence CP005428.2Saccharomyces cerevisiae chromosome XIII sequence CP005418.2Saccharomyces cerevisiae chromosome XIII sequence CP005408.2Saccharomyces cerevisiae chromosome XIII sequence CP005477.2Saccharomyces cerevisiae chromosome XIII sequence CP005417.2Saccharomyces cerevisiae chromosome XIII sequence CP005425.2Saccharomyces cerevisiae chromosome XIII sequence CP008265.1Saccharomyces cerevisiae chromosome XIII sequence CP008367.1Saccharomyces cerevisiae chromosome XIII sequence CP008554.1Saccharomyces cerevisiae chromosome XIII sequence CP008537.1Saccharomyces cerevisiae chromosome XIII sequence CP008520.1Saccharomyces cerevisiae chromosome XIII sequence CP008129.1Saccharomyces cerevisiae chromosome XIII sequence CP007993.1Saccharomyces cerevisiae chromosome XIII sequence CP005444.2Saccharomyces cerevisiae chromosome XIII sequence CP005434.2Saccharomyces cerevisiae chromosome XIII sequence CP005424.2Saccharomyces cerevisiae chromosome XIII sequence CP005404.2Saccharomyces cerevisiae chromosome XIII sequence CP005423.2Saccharomyces cerevisiae chromosome XIII sequence CP005422.2Saccharomyces cerevisiae chromosome XIII sequence CP005402.2Saccharomyces cerevisiae chromosome XIII sequence CP005421.2Saccharomyces cerevisiae chromosome XIII sequence CP005411.2Saccharomyces cerevisiae chromosome XIII sequence CP005420.2Saccharomyces cerevisiae chromosome XIII sequence CP005427.1Saccharomyces cerevisiae chromosome XIII sequence CP005416.1Saccharomyces cerevisiae chromosome XIII sequence CP008010.1Saccharomyces cerevisiae glucose-repressible alcohol KJ137141.1dehydrogenase II Saccharomyces cerevisiae chromosome XIII sequenceCP005475.2 Saccharomyces cerevisiae chromosome XIII sequence CP008401.1Saccharomyces cerevisiae chromosome XIII sequence CP008503.1Saccharomyces cerevisiae chromosome XIII sequence CP005398.2Saccharomyces cerevisiae chromosome XIII sequence CP005478.2Saccharomyces cerevisiae chromosome XIII sequence CP005437.2Saccharomyces cerevisiae chromosome XIII sequence CP005407.2Saccharomyces cerevisiae chromosome XIII sequence CP005454.2Saccharomyces cerevisiae chromosome XIII sequence CP005462.2Saccharomyces cerevisiae chromosome XIII sequence CP005461.2Saccharomyces cerevisiae chromosome XIII sequence CP005401.2Saccharomyces cerevisiae chromosome XIII sequence CP005396.1Saccharomyces cerevisiae chromosome XIII sequence CP005479.2Saccharomyces cerevisiae chromosome XIII sequence CP005469.2Saccharomyces cerevisiae chromosome XIII sequence CP005399.2Saccharomyces cerevisiae chromosome XIII sequence CP005397.2Saccharomyces cerevisiae chromosome XIII sequence CP005415.2Saccharomyces cerevisiae chromosome XIII sequence CP005395.2Saccharomyces cerevisiae chromosome XIII sequence CP008248.1Saccharomyces cerevisiae chromosome XIII sequence CP008333.1Saccharomyces cerevisiae chromosome XIII sequence CP008316.1Saccharomyces cerevisiae chromosome XIII sequence CP008299.1Saccharomyces cerevisiae chromosome XIII sequence CP008282.1Saccharomyces cerevisiae chromosome XIII sequence CP008231.1Saccharomyces cerevisiae chromosome XIII sequence CP008418.1Saccharomyces cerevisiae chromosome XIII sequence CP008384.1Saccharomyces cerevisiae chromosome XIII sequence CP008350.1Saccharomyces cerevisiae chromosome XIII sequence CP008486.1Saccharomyces cerevisiae chromosome XIII sequence CP008469.1Saccharomyces cerevisiae chromosome XIII sequence CP008435.1Saccharomyces cerevisiae chromosome XIII sequence CP008588.1Saccharomyces cerevisiae chromosome XIII sequence CP008571.1Saccharomyces cerevisiae chromosome XIII sequence CP008656.1Saccharomyces cerevisiae chromosome XIII sequence CP008639.1Saccharomyces cerevisiae chromosome XIII sequence CP008605.1Saccharomyces cerevisiae chromosome XIII sequence CP008214.1Saccharomyces cerevisiae chromosome XIII sequence CP008197.1

A lipase is an esterase that catalyzes the hydrolysis of fats, oils, andlipids. In one aspect, the gene that expresses lipase is isolated from abacterium. In a further aspect, the bacterium is a Micrococcus species,a Pseudomonas species, a Moraxella species, or an Acinetobacter species.In a further aspect, the gene that expresses lipase has SEQ ID NO. 6 orat least 70% homology thereof, at least 75% homology thereof, at least80% homology thereof, at least 85% homology thereof, at least 90%homology thereof, or at least 95% homology thereof. In a further aspect,the cellulose synthase is able to use mannose as a substrate instead ofor in addition to glucose. In one aspect, the gene that expresses lipasecan be positioned anywhere in the DNA construct disclosed herein. In oneaspect, the gene that expresses lipase is positioned 5′ (i.e., prior) tothe gene that expresses chitin synthase.

Other sequences expressing lipase or related or homologous genes can beidentified in a database such as, for example, GenBank. In one aspect,sequences useful herein include those with the GI numbers listed inTable 4:

TABLE 4 Lipase Genes Source Organism Sequence Description GI NumberMicrococcus sp. HL-2003 lipase gene AY268069.1 Pseudomonas sp. esterasegene M68491.1 Moraxella L1 lipase 1 X53053.1 A. calcoaceticuscarboxylesterase and X74839.1 peptidyl prolyl-cis- trans-isomeraseAcinetobacter sp. ADP1 genomic DNA CR543861.1 A. calcoaceticus esteraseX71598.1 Pseudomonas trivialis genomic DNA CP011507.1 Pseudomonasazotoformans genomic DNA CP019856.1 Pseudomonas genomic DNA LT629689.1extremaustralis Pseudomonas fluorescens genomic DNA CP005975.1Pseudomonas fluorescens genomic DNA CP010896.1 Pseudomonas fluorescensgenomic DNA AF228666.1 Pseudomonas simiae genomic DNA CP007637.1Pseudomonas fitiorescens genomic DNA AM181176.4 Pseudomonas Antarcticagenomic DNA CP015600.1 Pseudomonas fluorescens genomic DNA CP015639.1Pseudomonas fluorescens genomic DNA LT907842.1 Pseudomonas sp. NS1genomic DNA CP022960.1 Pseudomonas poae genomic DNA LT629706.1Pseudomonas poae genomic DNA CP004045.1 Pseudomonas rhodesiae genomicDNA LT629801.1 Pseudomonas trivialis genomic DNA LT629760.1 Pseudomonasazotoformans genomic DNA LT629702.1 Pseudomonas Antarctica genomic DNALT629704.1 Pseudomonas fluorescens genomic DNA CP012400.1 Pseudomonasazotoformans genomic DNA CP014546.1 Pseudomonas yamanorum genomic DNALT629793.1 Pseudomonas prosekii genomic DNA LT629762.1 Pseudomonaskoreensis genomic DNA CP014947.1 Pseudomonas libanensis genomic DNALT629699.1 Pseudomonas sp. GR 6-02 genomic DNA CP011567.1 Pseudomonasfluorescens genomic DNA CP014868.1 Pseudomonas fluorescens genomic DNACP011117.1 Pseudomonas fluorescens genomic DNA S69066.1 Pseudomonascedrina genomic DNA LT629753.1 Pseudomonas sp. bs2935 genomic DNALT629744.1 Pseudomonas fluorescens genomic DNA CP017296.1 Pseudomonassp. WCS374 genomic DNA CP007638.1 Pseudomonas fluorescens genomic DNACP003041.1 Pseudomonas corrugate genomic DNA LT629798.1 Pseudomonascorrugate genomic DNA CP014262.1 Pseudomonas mediterranea genomic DNALT629790.1 Pseudomonas tolaasii genomic DNA CP020369.1 Pseudomonasfluorescens genomic DNA CP015638.1 Pseudomonas fluorescens genomic DNACP015637.1 Pseudomonas sp. TKP genomic DNA CP006852.1 Syntheticconstruct carboxylesterase HM212419.1 Synthetic constructcarboxylesterase FJ213454.1 Pseudomonas sp. genomic DNA CP023969.1FDAARGOS 380 Pseudomonas synxantha genomic DNA LT629786.1 Pseudomonasorientalis genomic DNA LT629782.1 Pseudomonas sp. genomic DNA LN854573.1URMO17WK12: I11

In another aspect, said construct further includes (d) a promoter, (e) aterminator or stop sequence, (f) a gene that confers resistance to anantibiotic (a “selective marker”), (g) a reporter protein, or acombination thereof.

In another aspect, the DNA construct has the following geneticcomponents: (1) one or more promoters, (2) a gene that expresseszinc-related protein, (3) a gene that expresses alkaline phosphatase,(4) a gene that expresses alcohol dehydrogenase, and (5) one or moreterminators or stop sequences.

In an alternative aspect, the DNA construct has the following geneticcomponents: (1) one or more promoters, (2) a gene that expresseszinc-related protein, (3) a gene that expresses lipase, (4) a gene thatexpresses alkaline phosphatase, (5) a gene that expresses alcoholdehydrogenase, and (6) one or more terminators or stop sequences.

In one aspect, the construct includes a regulatory sequence. In afurther aspect, the regulatory sequence is already incorporated into avector such as, for example, a plasmid, prior to genetic manipulation ofthe vector. In another aspect, the regulatory sequence can beincorporated into the vector through the use of restriction enzymes orany other technique known in the art.

In one aspect, the regulatory sequence is a promoter. The term“promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence. In one aspect, the coding sequence tobe controlled is located 3′ to the promoter. In another aspect, thepromoter is derived from a native gene. In an alternative aspect, thepromoter is composed of multiple elements derived from different genesand/or promoters. A promoter can be assembled from elements found innature, from artificial and/or synthetic elements, or from a combinationthereof. It is understood by those skilled in the art that differentpromoters can direct the expression of a gene in different tissues orcell types, at different stages of development, in response to differentenvironmental or physiological conditions, and/or in different species.In one aspect, the promoter functions as a switch to activate theexpression of a gene.

In one aspect, the promoter is “constitutive.” A constitutive promoteris a promoter that causes a gene to be expressed in most cell types atmost times. In another aspect, the promoter is “regulated.” A regulatedpromoter is a promoter that becomes active in response to a specificstimulus. A promoter may be regulated chemically, such as, for example,in response to the presence or absence of a particular metabolite (e.g.,lactose or tryptophan), a metal ion, a molecule secreted by a pathogen,or the like. A promoter may also be regulated physically, such as, forexample, in response to heat, cold, water stress, salt stress, oxygenconcentration, illumination, wounding, or the like.

Promoters that are useful to drive expression of the nucleotidesequences described herein are numerous and familiar to those skilled inthe art. Suitable promoters include, but are not limited to, thefollowing: T3 promoter, T7 promoter, an iron promoter, and GAL1promoter. In one aspect, the promoter is the native GAL1 promoter foundin the plasmid pYES2. Variants of these promoters are also contemplated.The skilled artisan will be able to use site-directed mutagenesis and/orother mutagenesis techniques to modify the promoters to promote moreefficient function. The promoter may be positioned, for example, atabout 10-100 nucleotides from a ribosomal binding site. In anotheraspect, the promoter is positioned before the gene that expresseszinc-related protein, the gene that expresses alkaline phosphatase, thegene that expresses alcohol dehydrogenase, or any combination thereof

In one aspect, the promoter is a GAL1 promoter. In another aspect, theGAL1 promoter is native to the plasmid used to create the vector. Inanother aspect, a GAL1 promoter is positioned before the gene thatexpresses zinc-related protein, the gene that expresses alkalinephosphatase, and the gene that expresses alcohol dehydrogenase. Inanother aspect, the promoter is a GAL1 promoter obtained from or nativeto the pYES2 plasmid.

In another aspect, the regulatory sequence is a terminator or stopsequence. As used herein, a “terminator” is a sequence of DNA that marksthe end of a gene or operon to be transcribed. In a further aspect, theterminator is an intrinsic terminator or a Rho-dependent transcriptionterminator. As used herein, an intrinsic terminator is a sequencewherein a hairpin structure can form in the nascent transcript thatdisrupts the mRNA/DNA/RNA polymerase complex. As used herein, aRho-dependent transcription terminator requires a Rho factor proteincomplex to disrupt the mRNA/DNA/RNA polymerase complex. In one aspect,the terminator is a T7 terminator. In an alternative aspect, theterminator is a CYC1 terminator obtained from or native to the pYES2plasmid.

In a further aspect, the regulatory sequence includes both a promoterand a terminator or stop sequence. In a still further aspect, theregulatory sequence can include multiple promoters or terminators. Otherregulatory elements, such as enhancers, are also contemplated. Enhancersmay be located from about 1 to about 2000 nucleotides in the 5′direction from the start codon of the DNA to be transcribed, or may belocated 3′ to the DNA to be transcribed. Enhancers may be “cis-acting,”that is, located on the same molecule of DNA as the gene whoseexpression they affect.

In one aspect, when the vector is a plasmid, the plasmid can alsocontain a multiple cloning site or polylinker. In a further aspect, thepolylinker contains recognition sites for multiple restriction enzymes.The polylinker can contain up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or more than 20 recognition sites forrestriction enzymes. Further, restriction sites may be added, disabled,or removed as required, using techniques known in the art. In oneaspect, the plasmid contains restriction sites for any known restrictionenzyme such as, for example, HindIII, KpnI, SacI, BamHI, BstXI, EcoRI,BsaBI, NotI, XhoI, SphI, XbaI, ApaI, SalI, ClaI, EcoRV, PstI, SmaI,XmaI, SpeI, EagI, SacII, or any combination thereof. In a furtheraspect, the plasmid contains more than one recognition site for the samerestriction enzyme.

In one aspect, the restriction enzyme can cleave DNA at a palindromic oran asymmetrical restriction site. In a further aspect, the restrictionenzyme cleaves DNA to leave blunt ends; in an alternative aspect, therestriction enzyme cleaves DNA to leave “sticky” or overhanging ends. Inanother aspect, the enzyme can cleave DNA a distance of from 20 bases toover 1000 bases away from the restriction site. A variety of restrictionenzymes are commercially available and their recognition sequences, aswell as instructions for use (e.g., amount of DNA needed, precisevolumes of reagents, purification techniques, as well as informationabout salt concentration, pH, optimum temperature, incubation time, andthe like) are provided by enzyme manufacturers.

In one aspect, a plasmid with a polylinker containing one or morerestriction sites can be digested with one restriction enzyme and anucleotide sequence of interest can be ligated into the plasmid using acommercially-available DNA ligase enzyme. Several such enzymes areavailable, often as kits containing all reagents and instructionsrequired for use. In another aspect, a plasmid with a polylinkercontaining two or more restriction sites can be simultaneously digestedwith two restriction enzymes and a nucleotide sequence of interest canbe ligated into the plasmid using a DNA ligase enzyme. Using tworestriction enzymes provides an asymmetric cut in the DNA, allowing forinsertion of a nucleotide sequence of interest in a particular directionand/or on a particular strand of the double-stranded plasmid. Since RNAsynthesis from a DNA template proceeds from 5′ to 3′, usually startingjust after a promoter, the order and direction of elements inserted intoa plasmid can be especially important. If a plasmid is to besimultaneously digested with multiple restriction enzymes, these enzymesmust be compatible in terms of buffer, salt concentration, and otherincubation parameters.

In some aspects, prior to ligation using a ligase enzyme, a plasmid thathas been digested with a restriction enzyme is treated with an alkalinephosphatase enzyme to remove 5′ terminal phosphate groups. This preventsself-ligation of the plasmid and thus facilitates ligation ofheterologous nucleotide fragments into the plasmid.

In one aspect, different genes can be ligated into a plasmid in one pot.In this aspect, the genes will first be digested with restrictionenzymes. In certain aspects, the digestion of genes with restrictionenzymes provides multiple pairs of matching 5′ and 3′ overhangs thatwill spontaneously assemble the genes in the desired order. In anotheraspect, the genes and components to be incorporated into a plasmid canbe assembled into a single insert sequence prior to insertion into theplasmid. In a further aspect, a DNA ligase enzyme can be used to assistin the ligation process.

In another aspect, the ligation mix may be incubated in anelectromagnetic chamber. In one aspect, this incubation lasts for about1 minute, about 2 minutes, about 5 minutes, about 10 minutes, about 15minutes, about 20 minutes, about 30 minutes, or about 1 hour.

The DNA construct described herein can be part of a vector. In general,plasmid vectors containing replicon and control sequences that arederived from species compatible with the host cell are used inconnection with the hosts. The vector ordinarily carries a replicationsite as well as marking sequences that are capable of performingphenotypic selection in transformed cells. Plasmid vectors are wellknown and are commercially available. Such vectors include, but are notlimited to, pWLneo, pSV2cat, pOG44, pXT1, pSG, pSVK3, pBSK, pBR322,pYES, pYES2, pBSKII, pUC, and pUC19 vectors.

Plasmids are double-stranded, autonomously-replicating, genetic elementsthat are not integrated into host cell chromosomes. Further, thesegenetic elements are usually not part of the host cell's centralmetabolism. In bacteria, plasmids may range from 1 kilobase (kb) to over200 kb. Plasmids can be engineered to encode a number of useful traitsincluding the production of secondary metabolites, antibioticresistance, the production of useful proteins, degradation of complexmolecules and/or environmental toxins, and others. Plasmids have beenthe subject of much research in the field of genetic engineering, asplasmids are convenient expression vectors for foreign DNA in, forexample, microorganisms. Plasmids generally contain regulatory elementssuch as promoters and terminators and also usually have independentreplication origins. Ideally, plasmids will be present in multiplecopies per host cell and will contain selectable markers (such as genesfor antibiotic resistance) to allow the skilled artisan to select hostcells that have been successfully transfected with the plasmids (forexample, by growing the host cells in a medium containing theantibiotic.

In another aspect, the DNA construct includes a terminator. In a furtheraspect, the terminator is native to the vector in which the DNAconstruct is incorporated. In an alternative aspect, a terminator ispositioned after each gene of interest, in the 5′ to 3′ direction.

In one aspect, the vector encodes a selection marker. In a furtheraspect, the selection marker is a gene that confers resistance to anantibiotic. In certain aspects, during fermentation of host cellstransformed with the vector, the cells are contacted with theantibiotic. For example, the antibiotic may be included in the culturemedium. Cells that have not successfully been transformed cannot survivein the presence of the antibiotic; only cells containing the vector thatconfers antibiotic resistance can survive. Optimally, only cellscontaining the vector to be expressed will be cultured, as this willresult in the highest production efficiency of the desired gene products(e.g., proteins). Cells that do not contain the vector would otherwisecompete with transformed cells for resources. In one aspect, theantibiotic is tetracycline, neomycin, kanamycin, ampicillin, hygromycin,chloramphenicol, amphotericin B, bacitracin, carbapenam, cephalosporin,ethambutol, fluoroquinolones, isonizid, methicillin, oxacillin,vancomycin, streptomycin, quinolones, rifampin, rifampicin,sulfonamides, cephalothin, erythromycin, streptomycin, gentamycin,penicillin, other commonly-used antibiotics, or a combination thereof.

In certain aspects, the DNA construct can include a gene that expressesa reporter protein. The selection of the reporter protein can vary. Forexample, the reporter protein can be a yellow fluorescent protein, a redfluorescent protein, a green fluorescent protein, or a cyan fluorescentprotein. In one aspect, the reporter protein is a yellow fluorescentprotein and the gene that expresses the reporter protein has SEQ ID NO.4 or at least 70% homology thereto. The amount of fluorescence that isproduced by the biological device can be correlated to the amount of DNAincorporated into the microbial host cells. The fluorescence produced bythe device can be detected and quantified using techniques known in theart. For example, spectrofluorometers are typically used to measurefluorescence. The Examples provide exemplary procedures for measuringthe amount of fluorescence as a result of the expression of DNA.

FIGS. 1A and 1B provide non-limiting example of a DNA constructdescribed herein. In one aspect, the construct is a pYES2 plasmid havingfrom 5′ to 3′ the following genetic components in the following order:(a) a gene that expresses a zinc-related protein, (b) a gene thatexpresses an alkaline phosphatase, and (c) a gene that expresses analcohol dehydrogenase. In another aspect, the construct is a pYES2plasmid having from 5′ to 3′ the following genetic components in thefollowing order: (a) a GAL1 promoter, (b) a gene that expresseszinc-related protein, (c) a CYC1 terminator, (d) a GAL1 promoter, (e) agene that expresses alkaline phosphatase, (f) a CYC1 terminator, (g) aGAL1 promoter, (h) a gene that expresses alcohol dehydrogenase, and (i)a CYC1 terminator.

FIGS. 2A and 2B provide an additional non-limiting example of a DNAconstruct described herein. In one aspect, the construct is a pYES2plasmid having from 5′ to 3′ the following genetic components in thefollowing order: (a) a gene that expresses a zinc-related protein, (b) agene that expresses a lipase, (c) a gene that expresses an alkalinephosphatase, and (d) a gene that expresses an alcohol dehydrogenase. Inanother aspect, the construct is a pYES2 plasmid having from 5′ to 3′the following genetic components in the following order: (a) a gene thatexpresses zinc-related protein, (b) a CYC1 terminator, (c) a GAL1promoter, (d) a gene that expresses lipase, (e) CYC1 terminator, (f) aGAL1 promoter, (g) a gene that expresses alkaline phosphatase, (h) aCYC1 terminator, (i) a GAL1 promoter, (j) a gene that expresses alcoholdehydrogenase, and (k) a CYC1 terminator.

In another aspect, the construct comprises from 5′ to 3′ the followinggenetic components in the following order: (a) a gene that expresseszinc related protein having SEQ ID NO. 1 or at least 70% homologythereto; (b) a CYC1 terminator, (c) a GAL1 promoter, (d) a gene thatexpresses alkaline phosphatase having SEQ ID NO. 2 or at least 70%homology thereto; (e) a CYC1 terminator, (f) a GAL1 promoter, (g) a genethat expresses alcohol dehydrogenase having SEQ ID NO. 3 or at least 70%homology thereto, and (h) a CYC1 terminator.

In a further aspect, the construct comprises from 5′ to 3′ the followinggenetic components in the following order: (a) a gene that expresseszinc related protein having SEQ ID NO. 1 or at least 70% homologythereto; (b) a CYC1 terminator, (c) a GAL1 promoter, (d) a gene thatexpresses lipase having SEQ ID NO. 6 or at least 70% homology thereto,(e) a CYC1 terminator, (f) a GAL1 promoter, (g) a gene that expressesalkaline phosphatase having SEQ ID NO. 2 or at least 70% homologythereto; (h) a CYC1 terminator, (i) a GAL1 promoter, (j) a gene thatexpresses alcohol dehydrogenase having SEQ ID NO. 3 or at least 70%homology thereto, and (k) a CYC1 terminator.

In another aspect, the construct is a plasmid having from 5′ to 3′ thefollowing genetic components in the following order: (a) a GAL1promoter, (b) a gene that expresses a zinc-related protein having SEQ IDNO. 1 or at least 70% homology thereto, (c) a CYC1 terminator, (d) aGAL1 promoter, (e) a gene that expresses an alkaline phosphatase havingSEQ ID NO. 2 or at least 70% homology thereto, (f) a CYC1 terminator,(g) a GAL1 promoter, (h) a gene that expresses an alcohol dehydrogenasehaving SEQ ID NO. 3 or at least 70% homology thereto, and (i) a CYC1terminator.

In yet another aspect, the construct is a plasmid having from 5′ to 3′the following genetic components in the following order: (a) a gene thatexpresses a zinc-related protein having SEQ ID NO. 1 or at least 70%homology thereto, (b) a CYC1 terminator, (c) a GAL1 promoter, (d) a genethat expresses lipase having SEQ ID NO. 6 or at least 70% homologythereto, (e) a CYC1 terminator, (f) a GAL1 promoter, (g) a gene thatexpresses an alkaline phosphatase having SEQ ID NO. 2 or at least 70%homology thereto, (h) a CYC1 terminator, (i) a GAL1 promoter, (j) a genethat expresses an alcohol dehydrogenase having SEQ ID NO. 3 or at least70% homology thereto, and (k) a CYC1 terminator.

In a further aspect, the DNA construct has SEQ ID NO. 5 or at least 70%homology thereto.

In an alternative aspect, the DNA construct has SEQ ID NO. 7 or at least70% homology thereto.

Exemplary methods for producing the DNA constructs described herein areprovided in the Examples. Restriction enzymes and purificationtechniques known in the art can be used to assemble the DNA constructs.Backbone plasmids and synthetic inserts can be mixed together forligation purposes at different ratios ranging from 1:1, 1:2, 1:3, 1:4,and up to 1:5. In one aspect, the ratio of backbone plasmid to syntheticinsert is 1:4. After the vector comprising the DNA construct has beenproduced, the resulting vector can be incorporated into the host cellsusing the methods described below.

II. Biological Devices

In one aspect, a “biological device” is formed when a microbial cell istransfected with the DNA construct described herein. The biologicaldevices are generally composed of microbial host cells, where the hostcells are transformed with a DNA construct described herein.

In one aspect, the DNA construct is carried by the expression vectorinto the cell and is separate from the host cell's genome. In anotheraspect, the DNA construct is incorporated into the host cell's genome.In still another aspect, incorporation of the DNA construct into thehost cell enables the host cell to produce oxidized zinc. “Heterologous”genes and proteins are genes and proteins that have been experimentallyinserted into a cell that are not normally expressed by that cell. Aheterologous gene may be cloned or derived from a different cell type orspecies than the recipient cell or organism. Heterologous genes may beintroduced into cells by transfection or transformation.

An “isolated” nucleic acid is one that has been separated from othernucleic acid molecules and/or cellular material (peptides, proteins,lipids, saccharides, and the like) normally present in the naturalsource of the nucleic acid. An “isolated” nucleic acid may optionally befree of the flanking sequences found on either side of the nucleic acidas it naturally occurs. An isolated nucleic acid can be naturallyoccurring, can be chemically synthesized, or can be a cDNA molecule(i.e., is synthesized from an mRNA template using reverse transcriptaseand DNA polymerase enzymes).

“Transformation” or “transfection” as used herein refers to a processfor introducing heterologous DNA into a host cell. Transformation canoccur under natural conditions or may be induced using various methodsknown in the art. Many methods for transformation are known in the artand the skilled practitioner will know how to choose the besttransformation method based on the type of cells being transformed.Methods for transformation include, for example, viral infection,electroporation, lipofection, chemical transformation, and particlebombardment. Cells may be stably transformed (i.e., the heterologous DNAis capable of replicating as an autonomous plasmid or as part of thehost chromosome) or may be transiently transformed (i.e., theheterologous DNA is expressed for only a limited period of time).

“Competent cells” refers to microbial cells capable of taking upheterologous DNA. Competent cells can be purchased from a commercialsource, or cells can be made competent using procedures known in theart. Exemplary procedures for producing competent cells are provided inthe Examples.

The host cells as referred to herein include their progeny, which areany and all subsequent generations formed by cell division. It isunderstood that not all progeny may be identical due to deliberate orinadvertent mutations. A host cell may be “transfected” or“transformed,” which refers to a process by which exogenous nucleic acidis transferred or introduced into the host cell.

A transformed cell includes the primary subject cell and its progeny.The host cells can be naturally-occurring cells or “recombinant” cells.Recombinant cells are distinguishable from naturally-occurring cells inthat naturally-occurring cells do not contain heterologous DNAintroduced through molecular cloning procedures. In one aspect, the hostcell is a prokaryotic cell such as, for example, Escherichia coli. Inother aspects, the host cell is a eukaryotic cell such as, for example,the yeast Saccharomyces cerevisiae. Host cells transformed with the DNAconstruct described herein are referred to as “biological devices.”

The DNA construct is first delivered into the host cell. In one aspect,the host cells are naturally competent (i.e., able to take up exogenousDNA from the surrounding environment). In another aspect, cells must betreated to induce artificial competence. This delivery may beaccomplished in vitro, using well-developed laboratory procedures fortransforming cell lines. Transformation of bacterial cell lines can beachieved using a variety of techniques. One method involves calciumchloride. The exposure to the calcium ions renders the cells able totake up the DNA construct. Another method is electroporation. In thistechnique, a high-voltage electric field is applied briefly to cells,producing transient holes in the membranes of the cells through whichthe vector containing the DNA construct enters. Another method involvesexposing intact yeast cells to alkali cations such as, for example,lithium. In one aspect, this method includes exposing yeast to lithiumacetate, polyethylene glycol, and single-stranded DNA such as, forexample, salmon sperm DNA. Without wishing to be bound by theory, thesingle-stranded DNA is thought to bind to the cell wall of the yeast,thereby blocking plasmids from binding. The plasmids are then free toenter the yeast cell. Enzymatic and/or electromagnetic techniques canalso be used alone, or in combination with other methods, to transformmicrobial cells. Exemplary procedures for transforming yeast andbacteria with specific DNA constructs are provided in the Examples. Incertain aspects, two or more types of DNA can be incorporated into thehost cells. Thus, different metabolites can be produced from the samehost cells at enhanced rates.

III. Preparation of Oxidized Zinc

The biological devices described herein are useful in the production ofoxidized zinc. The oxidized zinc is any chemical species that includeszinc ions. For example the oxidized zinc can be a Zn⁺¹ or Zn⁺² species.In one aspect, the oxidized zinc can be an inorganic material such as,for example, ZnO. In another aspect, the oxidized zinc can be Zn⁺² withorganic groups or molecules bonded to the zinc ion through covalentbonds, electrostatic bonds, hydrogen bonding, Lewis acid/baseinteractions, or Vander Waals bonds. The organic groups can be smallmolecules or macromolecules such as proteins. The biological devicesdescribed herein can produce a composition composed of one or more zincion species.

Once the DNA construct has been incorporated into the host cell, thecells are cultured such that the cells multiply. A satisfactorymicrobiological culture contains available sources of hydrogen donorsand acceptors, carbon, nitrogen, sulfur, phosphorus, inorganic salts,and, in certain cases, vitamins or other growth-promoting substances.For example, the addition of peptone provides a readily-available sourceof nitrogen and carbon. Furthermore, the use of different types of mediaresults in different growth rates and different stationary phasedensities; stationary phase is where secondary metabolite productionoccurs most frequently. A rich media results in a short doubling timeand higher cell density at stationary phase. Minimal media results inslow growth and low final cell densities. Efficient agitation andaeration increase final cell densities.

In one aspect, host cells may be cultured or fermented by any methodknown in the art. The skilled practitioner will be able to select aculture medium based on the species and/or strain of host cell selected.In certain aspects, the culture medium will contain a carbon source. Avariety of carbon sources are contemplated, including, but not limitedto: monosaccharides such as glucose and fructose, disaccharides such aslactose or sucrose, oligosaccharides, polysaccharides such as starch, ormixtures thereof. Unpurified mixtures extracted from feedstocks are alsocontemplated as carbon sources, as are one-carbon substrates such ascarbon dioxide and/or methanol in the cases of compatible organisms. Thecarbon source utilized is limited only by the particular organism beingcultured.

Culturing or fermenting of host cells may be accomplished by anytechnique known in the art. In one aspect, batch fermentation may beconducted. In batch fermentation, the composition of the culture mediumis set at the beginning and the system is closed to future artificialalterations. In some aspects, a limited form of batch fermentation maybe carried out wherein factors such as oxygen concentration and pH aremanipulated, but additional carbon is not added. Continuous fermentationmethods are also contemplated. In continuous fermentation, equal amountsof a defined medium are continuously added to and removed from abioreactor. In other aspects, microbial host cells are immobilized on asubstrate. Fermentation may be carried out on any scale and may includemethods in which literal “fermentation” is carried out as well as otherculture methods that are non-fermentative.

In one aspect, the biological devices described herein are provided withan impure source of zinc or zinc oxide. In a further aspect, the impuresource of zinc or zinc oxide can be an ore, a recycled material, anenvironmental sample such as soil with a high zinc content, zinc metal,a zinc salt, or a combination thereof. In a still further aspect, thesource of zinc or zinc oxide can be in solution or suspension in wateror another solvent, including acidic or basic solutions, or can be inthe form of finely ground particles or in another solid form.

In one aspect, the method involves growing the biological devicesdescribed herein for a sufficient time to produce oxidized zinc from theimpure source of zinc or zinc oxide. The ordinary artisan will be ableto choose a culture medium and optimum culture conditions based on thebiological identity of the host cells.

In one aspect, the biological device can be exposed to UV radiation atwavelength of 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, or 400 nm,where any value can be a lower or upper end-point of a range (e.g., 250nm to 400 nm, 300 nm to 375 nm, etc.). The exposure to UV radiation canbe from 0.5 hours to 120 hours.

In certain aspects, after culturing the biological device to produce theoxidized zinc, the host cells of the device can be lysed with one ormore enzymes. For example, when the host cells are yeast, the yeastcells can be lysed with lyticase. In one aspect, the lyticaseconcentration can be 500, 600, 700, 800, 900, or 1000 μL per liter ofculture, where any value can be the lower or upper endpoint of a range(e.g. 500 to 900 μL, 600 to 800 μL, etc.).

In addition to enzymes, other components can be used to facilitate lysisof the host cells. In one aspect, chitosan can be used in combinationwith an enzyme to lyse the host cells. Chitosan is generally composed ofglucosamine units and N-acetylglucosamine units and can be chemically orenzymatically extracted from chitin, which is a component of arthropodexoskeletons and fungal and microbial cell walls. In certain aspects,the chitosan can be acetylated to a specific degree of acetylation inorder to enhance metabolite production. In one aspect, the chitosan isfrom 60% to about 100%, 70% to 90%, 75% to 85%, or is about 80%acetylated. The molecular weight of the chitosan can vary, as well. Forexample, the chitosan comprises about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 glucosamine unit and/orN-acetylglucosamine units. In another aspect, the chitosan includes 5 to7 glucosamine units and/or N-acetylglucosamine units. In one aspect, thechitosan can be added until a concentration of 0.0015, 0.0025, 0.0050,0.0075, 0.01, 0.015, 0.02, 0.03, 0.04, or 0.05% (v/v) is achieved in theculture, where any value can be a lower or an upper end-point of a range(e.g., 0.005 to 0.02%, 0.0075 to 0.015%, etc.). Still further in thisaspect, the chitosan is present at a concentration of 0.01%.

In a further aspect, the oxidized zinc can be collected, separated fromthe microbial cells (lysed or intact), and/or purified through anytechnique known in the art such as, for example, precipitation,centrifugation, filtration, or the like. The Examples provide anexemplary procedure for producing and purifying the oxidized zincdescribed herein.

In one aspect, compositions composed of the oxidized zinc with lysedand/or intact host cells can be used herein where it is not necessary toseparate the host cells and other components from the oxidized zinc.

IV. Applications of the Oxidized Zinc

In one aspect, the oxidized zinc produced herein can be used in theproduction of ceramic components, semiconductors, or electricalcomponents such as, for example, solar cells. Further in this aspect,the oxidized zinc produced herein may protect such components againstlightning or other voltage surges.

In an alternative aspect, the oxidized zinc produced herein can be usedin the production of glasses. In one aspect, the low coefficient ofexpansion of oxidized zinc can help glass materials resist thermaland/or mechanical shock. In a further aspect, glasses produced with theoxidized zinc described herein have high refractive indices and highthermal conductivity. In a still further aspect, inclusion of theoxidized zinc oxide herein in glass formulations reduces the fusionpoint of the glasses during melting. In one aspect, oxidized zincproduced herein can impart some or all of these properties to othermaterials in which the zinc oxide is included such as, for example,plastics, ceramics, glass, cement, rubber, lubricants, paints,ointments, adhesives, sealants, concrete, pigments, foods, batteries,fire retardants, and the like.

In a still further aspect, the oxidized zinc produced herein can be usedin cosmetic, pharmaceutical, or medical applications. In one aspect, theoxidized zinc has astringent properties and can be used in skincareproducts. In another aspect, the oxidized zinc can be used in sunscreencreams. In still another aspect, the oxidized zinc can be used as adietary supplement for humans or a micronutrient sample for livestockand/or other animals. In one aspect, the oxidized zinc can be applied tosoil in order to increase crop yield. In a still further aspect, theoxidized zinc can be used in wound healing applications such as, forexample, bandages, gauze, and other wound dressings, due to itsantifungal properties. In yet another aspect, the oxidized zinc can beused in dental cement or can be used as a precursor to producecomponents of dental cement.

In yet another aspect, the oxidized zinc described herein can beincorporated on the surface of or throughout various materials to impartdesired properties. In one aspect, when incorporated into paints, inks,and dyes, the oxidized zinc acts as a white pigment or as a brightener.In another aspect, the oxidized zinc may protect rubber, plastic, orother polymeric products such as, for example, outdoor furniture, fromultraviolet damage or may impart heat resistance to these items. In yetanother aspect, the oxidized zinc can act as a reinforcing agent and/orimpart abrasion resistance to objects and materials containing or coatedwith the oxidized zinc. In any of these applications, the oxidized zincmay aid in color retention due to its ultraviolet-absorbing properties.In a further aspect, the oxidized zinc can impart resistance tobacterial and fungal growth, including the growth of mildew and mold, toobjects coated with or formulated to contain the oxidized zinc.

In a further aspect, the oxidized zinc can be used as an activator inthe curing and/or vulcanization of rubber or latex products, or canimprove adhesion in adhesive products. Further in this aspect, theoxidized zinc produced herein can be used with stearic acid in thevulcanization process and can also protect rubber from colonization byfungi. In an alternative aspect, the oxidized zinc can be dissolved inan acid and used for an industrial process. Further in this aspect, theacid can be phosphoric acid and the resulting solution can be used forcoating or priming metal. Alternatively in this aspect, the acid can besulfuric acid and the resulting solution can be used for electroplating.

In one aspect, the oxidized zinc can be used as a precursor compound inthe manufacture of another zinc salt such as, for example, zincgluconate, zinc borate, zinc chloride, zinc dithiophosphate, zincchromate, zinc diacrylate, or another zinc salt.

In another aspect, the oxidized zinc produced herein may be useful inthe production of ceramics. In one aspect, the oxidized zinc can beincorporated into a ceramic glaze or frit. Further in this aspect, theoxidized zinc can affect the melting point and/or optical properties ofobjects coated with or formed from the oxidized zinc.

In another aspect, the extracts described herein can be applied to anymaterial that may benefit from a reduction in UV radiation. The exactformulation of the extract plus any carriers can be adjusted based onthe desired use. In one aspect, the extract is formulated with onlynon-toxic components if it is to be used on a human or animal or withanother microorganism, such as in a fermentation process or on anagricultural product. In another aspect, the extract can be mixed withother substances to provide UV-protective properties to the overallcomposition. In still another aspect, if coated on the material to beprotected, the extract itself can be covered with a further protectivecoating to project, for example, against mechanical wear and damage.

In the case when the extract is applied to the surface of an article, itcan be applied using techniques known in the art such spraying orcoating. In other aspects, the extract can be intimately mixed with asubstance or material that ultimately produces the article. For example,the extract can be mixed with molten glass so that the extract isdispersed throughout the final glass product.

In one aspect, the extract is formulated or applied in such a manner asto block approximately 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the UVradiation that encounters the extract, where any value can be a lowerand upper end-point of a range (e.g., 60% to 95%). In a further aspect,the extract can also be formulated to block these percentages ofparticular UV wavelengths, or, more generally, to block thesepercentages of UVA, UVB, or UVC radiation.

The extracts described herein can be used for a variety of purposes.These purposes include, but are not limited to, the following:

-   1. blocking UV radiation or other types of radiation;-   2. protecting human skin against damage and/or skin cancer induced    by UV radiation or other types of radiation;-   3. protecting against side effects of radiation used in cancer    treatments;-   4. protecting animals from deleterious effects of UV radiation or    other radiation;-   5. protecting plastic, fiberglass, glass, rubber, or other solid    surfaces from UV radiation or other radiation;-   6. providing a UV radiation screen or screen for other types of    radiation;-   protecting astronauts and/or other persons or organisms as well as    equipment during space trips;-   8. enhancement of industrial fermentation processes or other    processes requiring energy by allowing the use of UV radiation in    connection with the process to supply additional energy and thus to    increase the ultimate energy-requiring output of the cells without    substantially killing the fermenting organism;-   9. protection of experimentation, fermentation, biochemical, and/or    biological processes under the presence of UV radiation, for example    in extraterrestrial conditions such as on the moon or Mars; and-   10. protection of agricultural plants, particularly agricultural    plants in which the revenue-producing part of the plant is above    ground, such as fruits, vine vegetables, beans and peas, and leaf    vegetables.

In one particular embodiment, the extracts described herein can beapplied to an agricultural plant. In one aspect, the plant can be onethat produces fruit or vegetable, such as, for example, a watermelon ora tomato. Further in this aspect, the extract can be applied during atleast a part of the plant's growth to increase the amounts of one ormore nutrients of the fruit or vegetable, such as a vitamin, mineral, orother recommended dietary component. In one specific aspect, the amountof lycopene can be increased (which may be accompanied by a decrease incarotene or other less-valuable nutrients formed by competing pathways).In another aspect, the amount of a flavor-enhancing component, such asglucose, can be increased. Further in this aspect, an increase inglucose can help protect against water loss.

In one aspect, the extract can be applied for about 25%, 50%, 75%, 90%,95%, or 99% of the fruit or vegetable's on-plant life, where theon-plant life includes the time span from the formation of a separatebody that will constitute the fruit or vegetable (in some aspects,excepting flowers) until the fruit or vegetable is harvested. In oneaspect, the extract can be first applied when the fruit or vegetable issufficiently large to no longer be substantially protected from UVradiation by leaves. In another aspect, the extract can first be appliedfive days, one week, or two weeks prior to harvest. Further in thisaspect, application at this later stage can be particularly useful withfruits or vegetables in which an increase in a nutrient orflavor-enhancing component can be obtained by protecting the fruit orvegetable from UV radiation later in its on-plant life.

In one aspect, the extract can be applied once or multiple times to eachfruit or vegetable. In another aspect, it can be applied weekly, or itcan be reapplied after the fruit or vegetable is exposed to rain orafter a turning process. In another aspect, the agricultural plant canbe another food crop that grows above ground and is exposed to naturalUV radiation, wherein the agricultural product produced can be a fruit,leaf, seed, flower, grain, nut, stem, vegetable, or mushroom.

In another aspect, it is desirable for agricultural plants that do notproduce parts typically consumed by humans to be protected from UVirradiation. In a further aspect, these other agricultural plants canincludes sources of fibers such as, for example, cotton and linen(flax), of cork, of wood or lumber, of feedstocks for producing ethanolor biodiesel (including, but not limited to, sugar beet, sugarcane,cassava, sorghum, corn, wheat, oil palm, coconut, rapeseed, peanut,sunflower, soybean, and the like), of animal feedstocks or fodder, or ofdecorative or horticultural plants.

In one aspect, any part of the plant can be coated, including, but notlimited to, the part of the plant that is collected during harvest. Inan alternative aspect, the harvested part of the plant is not coated,but another part can be coated with the extracts disclosed herein. Inaddition to the aspects already described, in one aspect, coating aplant with the extracts described herein can prolong the life of theplant, increase production capacity of a desired product, can increasethe growth rate of the plant relative to an untreated plant of the sametype, can increase production of a desired metabolite that mightotherwise decrease due to UV-induced stress, can increase yield of acrop of such plants, and the like.

In a further aspect, application can be accomplished with a commercialsprayer. In another aspect, application can be only on the upperportions of the fruit or vegetable, which are exposed to substantiallygreater amounts of UV radiation than the lower portions of the fruit orvegetable.

In another aspect, provided herein is a pharmaceutical compositioncontaining the extracts produced by the biological devices describedherein. In one aspect, the pharmaceutical composition can be applied toa subject, wherein the subject is exposed to radiation. In one aspect,the radiation is applied as a strategy to treat cancer. In anotheraspect, the pharmaceutical composition is used to preventradiation-induced cellular and DNA damage. In another aspect, dosageranges of the extract in the pharmaceutical composition can vary from0.01 g extract/mL of pharmaceutical composition to 1 g extract/mL ofpharmaceutical composition, or can be 0.01, 0.02, 0.025, 0.05, 0.075, or1 g extract/mL of pharmaceutical composition. In an alternative aspect,provided herein is a cosmetic composition containing the extractsproduced by the biological devices described herein. Further in thisaspect, the cosmetic composition can be a cleanser, lotion, cream,shampoo, hair treatment, makeup, lip treatment, nail treatment, orrelated composition. In still a further aspect, the compositionscontaining the extracts can have both pharmaceutical and cosmeticapplications. In yet another aspect, the compositions containing theextracts can be used in veterinary medicine.

In one aspect, the oxidized zinc produced herein can be formulated,along with iron (III) oxide, as calamine lotion to relieve itch. Inanother aspect, the oxidized zinc can be mixed with eugenol and used asa prosthodontic. In another aspect, the oxidized zinc can be added as aningredient to baby powder, barrier creams, diaper rash treatments,anti-dandruff shampoos, antiseptic ointments, burn treatment creams,hemorrhoid creams, and the like.

In another aspect, the oxidized zinc can be milled or ground into fineparticles and incorporated into materials and compositions for itsdesirable deodorizing or antibacterial properties for use on the body,on surfaces, in healthcare settings, and the like.

The cosmetic compositions can be formulated in any physiologicallyacceptable medium typically used to formulate topical compositions. Thecosmetic compositions can be in any galenic form conventionally used fora topical application such as, for example, in the form of dispersionsof aqueous gel or lotion type, emulsions of liquid or semi-liquidconsistency of the milk type, obtained by dispersing a fatty phase in anaqueous phase (0/W) or vice versa (W/O), or suspensions or emulsions ofsoft, semi-solid or solid consistency of the cream or gel type, oralternatively multiple emulsions (W/O/VV or O/W/O), microemulsions,vesicular dispersions of ionic and/or non-ionic type, or wax/aqueousphase dispersions. These compositions are prepared according to theusual methods.

The cosmetic compositions can also contain one or more additivescommonly used in the cosmetics field, such as emulsifiers,preservatives, sequestering agents, fragrances, thickeners, oils, waxesor film-forming polymers. In one aspect, in any of the above scenarios,the pharmaceutical, cosmetic, or veterinary composition also includesadditional UV-protective compounds or UV-blocking agents such as, forexample, zinc oxide, titanium dioxide, carotenoids, oxybenzone,octinoxate, homosalate, octisalate, octocrylene, avobenzone, or acombination thereof.

In one aspect, the composition is a sunscreen. A sunscreen can beformulated with any of the extracts produced herein. In addition to theextract, the sunscreen in certain aspects can be formulated with one ormore UV-protective compounds or UV-blocking agents listed above. Thesunscreen can be formulated as a paste, lotion, cream, aerosol, or othersuitable formulations for topical use. In certain aspects, the sunscreencan be formulated as a transparent composition. In one aspect, whenincluded in a sunscreen, the oxidized zinc produced herein can blockboth UVA (320-400 nm) and UVB (280-320 nm) rays. In a still furtheraspect, when incorporated into a sunscreen or skincare or cosmeticproduct, the oxidized zinc is non-irritating, non-allergenic, andnon-comedogenic.

In one aspect, the cosmetic composition can be a film composed of theextracts produced herein that can be directly applied to the skin. Forexample, the film can be composed of a biocompatible material such as aprotein or oligonucleotide, where the extract is coated on one or moresurfaces of the film or, in the alternative dispersed throughout thefilm. For example, the film can be composed of DNA. In this application,the films can be used as a wound covering and provide protection from UVphotodamage. The films can also be prepared so that they are opticallytransparent. Here, it is possible to view the wound without removing thecovering and exposing the wound. The films can also include othercomponents useful in cosmetic applications such as, for example,compounds to prevent or reduce wrinkles.

In one aspect, the pharmaceutical, cosmetic, or veterinary compositionsdescribed herein are applied to subjects. In one aspect, the subject isa human, another mammal, or a bird. In a further aspect, the mammal is apet such as a dog or cat or is livestock such as horses, goats, cattle,sheep, and the like. In an alternative aspect, the bird is a pet bird oris poultry such as, for example, a chicken or turkey. In any of theseaspects, the compositions can be applied to skin, fur, feathers, wool,hooves, horns, or hair as appropriate and applicable.

In another aspect, provided herein is a paint, dye, stain, or inkcontaining the UV-protective and/or UV-resistant extract disclosedherein. In one aspect, there are several benefits to having a paint thatis resistant to UV irradiation. In a further aspect, imparting UVresistance to a paint slows or stops photodegradation, bleaching, orcolor fading. In another aspect, a paint with UV resistance preventschemical modification of exposed paint surfaces. Further in this aspect,chemical modification of exposed paint surfaces includes change infinish, structural changes in binders, flaking, chipping, and the like.In one aspect, the paint provided herein resists these changes.

In still another aspect, provided herein is an article coated with theextracts disclosed herein. In one aspect, the article is made of glass,plastic, metal, wood, fabric, or any combination thereof. In one aspect,the article is a construction material such as, for example, steel,concrete or cement, brick, wood, window or door glass, fiberglass,siding, wallboard, a flooring material, masonry, mortar, grout, stone,artificial stone, stucco, shingles, roofing materials, and the like. Inan alternative aspect, the material is an aeronautical or aerospacematerial such as, for example, the metal or metal alloy body of anaircraft or spacecraft, paint on the body of an aircraft or spacecraft,glass windows on an aircraft or spacecraft, carbon fiber composite,titanium or aluminum, a ceramic heat absorbing tile, and the like. Instill another aspect, the article is a fabric article such as, forexample, clothing, drapes, outdoor upholstery, a tent or outdoorpavilion, a flag or banner, or the like. In another aspect, the extractcan be applied to the article to fine artwork, solid pieces (e.g.,vases), and historical documents in order to preserve them. In anotheraspect, the extract can be applied to outdoor signs such as highwaybillboards and advertising.

In other aspects, the extract can be incorporated within or throughoutthe article. In one aspect, the extract can be mixed with molten glassto produce glass article that are UV resistant such as, for example,sunglasses, car windshields, window glass, and eyeglasses. In anotheraspect, the glass article can be a bottle for storing a beverage or foodcontainer in order to increase the shelf-life of the beverage or food.It is contemplated that the extract can be applied externally to theglass articles as well.

In another aspect, the extract can be mixed with fiberglass or plasticsin order to reduce negative effects to aircraft, watercraft, boats, jetskis, decking, house siding, motor homes, sunroofs, and moon roofs thatare constantly exposed to UV radiation. It is contemplated that theextract can be applied externally to the fiberglass or plastic articlesas well.

In another aspect, the extract can be mixed with rubber, silicon, orlatex used to make a variety of articles such as water hoses, tires, andthe like. It is contemplated that the extract can be applied externallyto the rubber, silicone, or latex articles as well.

In another aspect, the extract can be mixed with foams used to make avariety of articles such as automotive dashboard padding, seat cushions,and the like. It is contemplated that the extract can be appliedexternally to the foam articles as well.

In another aspect, the extracts described herein can be incorporatedinto an optical film. In one aspect, the extract is applied to at leastone surface of the film. In another aspect, the extract can be dispersedthroughout the film. The film can be transparent, translucent or opaque.The film can be composed of, but not limited to, polyolefin resin, suchas polyethylene (PE) or polypropylene (PP); polyester resin, such aspolyethylene terephthalate (PET); polyacrylate resin, such as polymethyl(meth)acrylate (PMMA); polycarbonate resin; polyurethane resin or amixture thereof. The optical film can be applied to any substrate whereit is desirable to reduce or prevent UV exposure or damage. For example,the optical film can be applied to windows to reduce or prevent UVradiation from entering a structure (e.g., building, vehicle, etc.).

In another aspect, provided herein is a method of reducing or preventingthe exposure of an item to UV radiation by applying the extractsdescribed herein to the item or incorporating the extractwithin/throughout the article. Further in this aspect, “reducing” isdefined relative to an untreated control. That is, if two like items areexposed to equal amounts of UV radiation for an equal amount of time,but one has been treated with the UV-resistant extracts and the otherhas not, and some objective response is measured (e.g., color fading,structural degradation, plant size or yield, etc.), the treated itemwill appear to have been exposed to a lower amount of UV (for example,the color of the treated item will have faded less and will remaincloser to the original, or a treated plant will appear larger and morevigorous and will have a greater yield, etc.). In some aspects,treatment with the extracts disclosed herein will prevent UV exposurefrom occurring. As used herein, “prevent” indicates that a treated itemwill not be affected, changed, or altered by UV exposure.

In one aspect, the extract blocks from 50% to 100% of UV radiation fromcontacting the item. Further in this aspect, the extract blocks at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of UVradiation from contacting the item. In another aspect, the extractblocks from 50% to 100% of longwave UV radiation from contacting theitem. Further in this aspect, the extract blocks at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of longwave UV radiationfrom contacting the item. In one aspect, the extract blocks from 50% to100% of shortwave UV radiation from contacting the item. Further in thisaspect, the extract blocks at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99%, or 100% of shortwave UV radiation from contactingthe item.

Depending upon the application, the extract can prevent or reduce damagecause by UV radiation from limited to extended periods of time. Byvarying the amount of extract that is applied as well as the number oftimes the extract is applied, the degree of UV protection can be varied.In certain aspects, it may be desirable for the article to be protectedfrom UV damage for a short period of time then subsequently biodegrade.

In another aspect, the extracts produced herein can be used to reduce orprevent the growth of barnacles on boats and other water vehicles. Inone aspect, the extract can be admixed with a paint that is typicallyapplied to water vehicles, where the paint also includes chitosan. Inone aspect, the chitosan can be acetylated to a specific degree ofacetylation in order to enhance tissue growth during culturing as wellas metabolite production. In one aspect, the chitosan is from 60% toabout 100%, 70% to 90%, 75% to 85%, or about 80% acetylated. In oneaspect, chitosan isolated from the shells of crab, shrimp, lobster,and/or krill is useful herein. The molecular weight of the chitosan canvary, as well. For example, the chitosan comprises about 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 glucosamine unitsand/or N-acetylglucosamine units. In another aspect, the chitosanincludes 5 to 7 glucosamine units and/or N-acetylglucosamine units.

In another aspect, the extracts produced herein can be formulated as apharmaceutical composition for increasing the zinc levels in a subject.Maintaining zinc levels in a subject is desirable for promoting goodhealth. In certain diseases, zinc levels are decreased significantly.For example, in certain cancers such as, for example, hepatocellularcarcinoma (HCC), the patient has a significant decrease in zinc levels.The extracts described herein can be formulated such that whenadministered to the subject increase zinc levels to a normal and healthyvalue. The extracts can be administered to a subject that is undergoingchemotherapy.

The extracts described herein can be formulated in any excipient thebiological system or entity can tolerate to produce pharmaceuticalcompositions. Examples of such excipients include, but are not limitedto, water, aqueous hyaluronic acid, saline, Ringer's solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Nonaqueous vehicles, such as fixed oils, vegetable oilssuch as olive oil and sesame oil, triglycerides, propylene glycol,polyethylene glycol, and injectable organic esters such as ethyl oleatecan also be used. Other useful formulations include suspensionscontaining viscosity enhancing agents, such as sodiumcarboxymethylcellulose, sorbitol, or dextran. Excipients can alsocontain minor amounts of additives, such as substances that enhanceisotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosol, cresols, formalin and benzyl alcohol.In certain aspects, the pH can be modified depending upon the mode ofadministration. For example, the pH of the composition is from about 5to about 6, which is suitable for topical applications. Additionally,the pharmaceutical compositions can include carriers, thickeners,diluents, preservatives, surface active agents and the like in additionto the compounds described herein.

In one aspect, the extract can also be injected parenterally eitherintravenously, subcutaneously, intramuscularly, intradermally,intranasally, or intrathecally. For example, the extract can beadministered rectally by an enema, suppository, catheter, needlelesssyringe, or bulb syringe. In other aspects, the extract can beformulated for oral administration in the form of a beverage, lozenge,tablet, capsule, or any other suitable medium for oral administration.

In one aspect, the oxidized zinc produced herein can be formulated asnanoparticles. In this aspect, oxidized zinc does not appear whiteagainst the skin when used in cosmetic applications. In a furtheraspect, oxidized zinc nanoparticles may help contribute to theantibiotic activity of ciprofloxacin and similar drugs by interferingwith the action of microbial proteins. In a still further aspect,oxidized zinc nanoparticles may be more difficult for bacteria todevelop resistance to than other antimicrobial ingredients.

In another aspect, the oxidized zinc produced herein can be a componentof cigarette filters, or can be added to foods, beverages, functionalfoods, vitamins, and supplements as a source of dietary zinc.

In one aspect, the oxidized zinc produced herein can be incorporatedinto pigments including oil paints and mineral makeup, or can be used asa paper coating. In still another aspect, the oxidized zinc can be usedas an anticorrosive coating for metals such as, for example, galvanizediron, or in nuclear reactors. In one aspect, paints and coatings thatinclude the oxidized zinc produced herein can be flexible andlong-lasting in the environment. In still another aspect, the oxidizedzinc produced herein can reduce photo-yellowing of plastics (such aspoly carbonate) and other materials.

In still another aspect, the oxidized zinc produced herein can be usedin a semiconductor such as one that is n-type doped with aluminum orgallium. In an alternative aspect, the oxidized zinc produced herein canbe used in electrical elements such as laser diodes, LEDs, fieldemitters, electrodes (when doped with aluminum), liquid crystaldisplays, transparent thin-film transistors, ferromagnets (incombination with Mn, Fe, Co, V, or another magnetic ion), piezoelectricdevices, anodes in lithium ion batteries, and/or sensors for electriccurrent or hydrogen gas.

In an alternative aspect, the oxidized zinc can be used in an industrialprocess such as pretreatment of natural gas to remove hydrogen sulfide.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, and methods described and claimed herein aremade and evaluated, and are intended to be purely exemplary and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperatures, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. or is at ambienttemperature, and pressure is at or near atmospheric. Numerous variationsand combinations of reaction conditions (e.g., component concentrations,desired solvents, solvent mixtures, temperatures, pressures, and otherreaction ranges and conditions can be used to optimize the productpurity and yield obtained from the desired process. Only reasonable androutine experimentation will be required to optimize such processes andconditions.

Example 1: Preparation of DNA Construct for Production of Oxidized Zinc

The DNA construct was composed of genetic components described hereinand assembled in plasmid vectors (e.g., pYES2). Sequences of genesand/or proteins with desired properties were identified in GenBank;these included a zinc-related protein gene, an alkaline phosphatase, andan alcohol dehydrogenase gene. Other genetic parts were also obtainedfor inclusion in the DNA constructs including, for example, promotergenes (e.g., GAL1 promoter), reporter genes (e.g., yellow fluorescentreporter protein), and terminator sequences (e.g., CYC1 terminator).These genetic parts included restriction sites for ease of insertioninto plasmid vectors.

The cloning of the DNA construct into the biological devices wasperformed as follows. Sequences of individual genes were amplified bypolymerase chain reaction using primers that incorporated restrictionsites at their 5′ ends to facilitate construction of the full sequenceto be inserted into the plasmid. Genes were then ligated using standardprotocols to form an insert. The plasmid was then digested withrestriction enzymes according to directions and using reagents providedby the enzyme's supplier (Promega). The complete insert, containingrestriction sites on each end, was then ligated into the plasmid.Successful construction of the insert and ligation of the insert intothe plasmid were confirmed by gel electrophoresis.

PCR was used to enhance DNA concentration using a Mastercycler Personal5332 ThermoCycler (Eppendorf North America) with specific sequenceprimers and the standard method for amplification (Sambrook, J., E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,2^(nd) ed., Vol. 1, Cold Spring Harbor Laboratory Press: Cold SpringHarbor, N.Y.). Digestion and ligation were used to ensure assembly ofDNA synthesized parts using restriction enzymes and reagents (PCR mastermix of restriction enzymes: XhoI, KpnI, XbaI, EcoRI, BamHI, and HindIII,with alkaline phosphatase and quick ligation kit, all from Promega). DNAwas quantified using a NanoVue spectrophotometer (GE Life Sciences) anda standard UV/Visible spectrophotometer using the ratio of absorbancesat 260 nm versus 280 nm. In order to verify final ligations, DNA wasvisualized and purified via electrophoresis using a Thermo EC-150 powersupply.

The DNA construct was made with gene parts fundamental for expression ofsequences such as, for example, ribosomal binding sites, native andconstitutive promoters, reporter genes, and transcriptional terminatorsor stops. Backbone plasmids and synthetic inserts can be mixed togetherfor ligation purposes at different ratios ranging from 1:1, 1:2, 1:3,1:4, and up to 1:5. In one aspect, the ratio of backbone plasmid tosynthetic insert is 1:4. The DNA constructs in FIG. 1 was assembledusing the techniques above.

After the vector comprising the DNA construct has been produced, theresulting vector can be incorporated into the host cells using themethods known in the art (e.g., Gietz, R. D. and R. H. Schiestl, 2007,Nature Protocols, “Quick and easy yeast transformation using the LiAc/SScarrier DNA/PEG method,” Vol. 2, 35-37, doi:10.1038/nprot.2007.14). Insome instances, competent yeast cells (strain INVSc1) were purchasedfrom Invitrogen, Inc. and transformed with a kit from Sigma Aldrich,Inc.

From a plate of transformed cells (SC dropout plate deficient inuracil), four clones were selected and processed for full-length DNAsequencing. A clone with 100% DNA sequence accuracy was selected forfurther processing and used to obtain a high concentration of theplasmid construct at a mid-scale plasmid purification level.

Example 2: Growth and Induction of the Yeast-Zinc Device

A small sample of the biological devices described herein (S. cerevisiaetransformed with the constructs in FIG. 1A-B) was mixed with 3-5 mL ofyeast malt (YM) for growing at 30° C., overnight. Growth and productionof the metabolites were induced as follows. 1 mL of the devices grownovernight were taken into 1 L of yeast malt medium containing 2%raffinose and incubated at 30° C. for 2-4 hours until the growth of theculture reached 0.6-0.6 optical density. Galactose sugar (1%) was thenadded to the above culture, which was then incubated for at least 48-72hours at 30° C.

Example 3: Extraction of Anti UV Metabolites and Compounds from theYeast-Zinc Device

After 48 hours, the culture from Example 2 was treated with lyticase(240 μL/L) for 24 hours. The culture was then centrifuged at 9000 rpmfor 15 minutes to obtain a pellet. The pellet was mixed with sterilizeddistilled water at a ratio of 1 g pellet/100 mL water. This mixture wassubjected to a protocol of sonication at alternating periods of 30seconds on, 15 seconds off, for 2 minutes at 60% of wavelength amplitude(QSONICA Sonicator, Newtown, Conn.); this procedure was repeated twice.The supernatant from the above sonication was centrifuged to discarddead cells and debris, then filtered through a 0.45 μm pore size filter.The filtrate was then used for further anti-UV tests.

Example 4: Electrochemical Analysis

Zinc oxide nanopowder with a particle size of <100 nm was used in thefollowing experiments. The nanopowder was partially soluble in water (pH7) but completely soluble in water with nitric acid or hydrochloric acidat pH 2. For pH values 3 or greater, a precipitate was observed (FIG.7A). For quantification of zinc by voltammetry, nitric acid was added tofacilitate the availability in solution.

The pH of the extracts disclosed herein was approximately 6.89, with thesoluble solids representing 0.0027 g/mL of device extract. At pH valuesof less than 3, a precipitate was observed, whereas appearance of theextracts was translucent at pH 10 (FIG. 7B).

0.05 g of ZnO nanopowder was mixed with 5 mL of HNO₃ (65%) and 1 mL of5M HCl and water was added to 50 mL. The solution was boiled for 10 min.An additional 5 mL of HNO₃ (65%) and 2 mL of 5M HCl were added and thesamples were brought to a final volume of 100 mL with water to make aclear solution (FIG. 7C).

To determine the amount of device extract needed to obtain 0.05 g ofsolids, the following equation was used:

0.05 g×0.0027 g/mL=18.5 mL

Thus, 18.5 mL of device extract were measured into a flask. 5 mL of HNO₃(65%) and 2 mL of HCl (5M) were added and deionized water was added toobtain a total volume of 50 mL. This solution was boiled for 10 min andbrought to a final volume of 100 mL in the same manner as for the zincoxide nanopowder samples (FIG. 7D).

Voltammetric measurements were conducted on a Voltamperimeter 797 VAComputrance (Metrohm) using 797 VA Computrance v. 1.3.2 software. Zinc(II) concentration was determined and quantified for experimentalsamples using ZnO nanopowder as the reference pattern. Samples wereprepared with 100 μL of sample in 10 μL of deionized water and 500 mL ofbuffer (ammonium acetate, pH 4.6). Instrumental parameters are providedin Table 5:

TABLE 5 Instrumental Parameters for Zinc Determination Working electrodeHMDE Stirrer speed 2000 rpm Mode DP Drop size 4 Calibration Standardaddition Purge time 300 s Deposition potential −1.15 V Deposition time60 s Equilibration time 5 s Pulse amplitude 0.05 V Start potential(condition cycles) −1.2 V End potential −0.1 V Start potential (sweep)−1.15 V End potential −0.3 V Voltage step 0.0059 V Voltage step time 0.4s Sweep rate 0.0149 V/s Peak potential Zn 0.96 V

Calculations were performed according to a protocol provided by themanufacturer. The voltamperimeter measured the intensity of current inamperes versus the registered voltage. The zinc peak is specificallydetected at −960 mV. Standard additions are made manually followingmeasurement of the initial sample. Sample intensity graphs for zincnanopowder in nitric acid and the zinc device extract without digestioncan be seen in FIGS. 8A and 8B, respectively. Device extracts werefiltered with a 0.2 μm nylon filter prior to measurement.

For quantification, calibration curves were constructed using a pureanalytical zinc standard solution (1 g/L, Panreac). Standard curves weredetermined for the analytical solution (FIG. 9A), zinc oxide in nitricacid (FIG. 9B), digested zinc nanopowder (FIG. 9C), the zinc deviceextract (FIG. 9D), a lower concentration of the unfiltered zinc deviceextract (FIG. 9E), and the digested zinc device extract filtered througha 0.2 μm nylon filter (FIG. 9F). Results from the voltammetricmeasurements are presented in Table 6:

TABLE 6 Voltammetric Measurements Zinc concentration Standard Correction(mg/L) deviation coefficient ZnO nanopowder 1070.198 ±108.351 0.9714Diluted digestion of 506.34 ±60.247 0.9362 ZnO nanopowder Concentratedzinc 11.05 ±0.156 0.9608 device extract Diluted zinc device 4.050 ±0.1050.9602 extract Digestion filtered 61.16 ±0.156 0.9696 (Nylon 0.2 μm)ofZinc device extract (diluted)

Based on measurements of the concentrated zinc device, an initial volumeof 1 L will result in 11.05 mg/L of zinc. For a filtered digestion ofthe diluted zinc device extract, an initial volume of 1 L of extractwill result in 61.16 mg/L of zinc. The zinc from the nanopowder and thedevice extracts was oxidized (Zn²⁺).

Example 6: Chemical Analysis

Zinc concentration was also determined by complexometric titration withEDTA. Eriochrome black was used as indicator with a color change fromblue to red (violet) to determine the endpoint of the titration. 0.05 gof zinc nanopowder sample was dissolved with 500 μL HCl with a finalsample volume of 25 mL. 5 mL of this solution was mixed with 2 mL of anammonium buffer (pH 9.5) and eriochrome black and 0.02M EDTA was addeduntil the solution turned blue at which point the titration was stopped.For zinc device extracts, 18.5 mL samples were used. 500 μL of 5M HClwere added to the extract samples and diluted with deionized water to 25mL, and then the titration was performed. Biological extract sampleswere filtered with a 0.2 μm nylon filter to reduce viscosity in order toperform the titrations. Results of Zn²⁺ determination are presented inTable 7:

TABLE 7 Determination of Zinc Concentration by Complexometric TitrationConcentration Standard (mg/L) deviation C.V. Zinc nanopowder 2124.836±29.921 1.41% Device extracts 22.675 ±6.478 28.57%

The components present in the extract were further evaluated. Theextract was evaporated in a water bath and reduced from an initialvolume of 60 mL to a final volume of 7 mL. This solution wasfractionated by a solid phase column manufactured with RP-18 (40-63 μm)from Merck. The stationary phase was activated with the initial water(0.1% TFA) phase, approximately 5 volumes of the stationary phase werepassed for a total of 125 mL. The concentrated extract solution wasadded followed by the addition of different solvents, with acetonitrilebeing added in 10%. A total of 11 fractions of 50 mL each were obtainedusing the following solvents: 1) Water (0.1% TFA). 2) 10% ANC: Water(0.1% TFA). 3) 20% ANC: Water (0.1% TFA). 4) 30% ACN: Water (0.1% TFA).5) 40% ACN: Water (0.1% TFA). 6) 50% ACN: Water (0.1% TFA). 7) 60% ACN:Water (0.1% TFA). 8) 70% ACN: Water (0.1% TFA). 9) 80% ACN: Water (0.1%TFA). 10) 90% ACN: Water (0.1% TFA). 11) 100% ACN.

TLC chromatography was performed with the first fraction collected fromthe column. The sample was concentrated by evaporation and applied tofour TLC RP-18 plates (10×10 cm) 4 plates. Subsequently, three bands(samples 1, 2, and 3) were cut from each plate and scraped from theplate. Additionally, TLC chromatography was performed with the sixthfraction collected from the column (sample 4). In order to extract thezinc compound from the stationary phase of each band, water (0.1% TFA)90:10 methanol was added to the scraping dust and it was taken to anultrasonic bath for 20 minutes 2 times each. Then each fraction wasconcentrated by evaporation and observed with the UV-lamp (365 nm). Theamount of oxidized zinc present in each sample was determined byelectrochemical analysis as described above with the results provided inTable 8. Thus, the extract produced by the zinc device produced multipleoxidized zinc species.

TABLE 8 Sample Result [mg/L] Std. Dev. Corr. Coeff. 1 0.593 ±0.0210.91789 2 3.835 ±0.087 0.93781 3 3.166 ±0.087 0.90946 4 5.002 ±0.1190.93693

Example 7: Use of the Yeast-Zinc Device for UV Protection of BacterialCultures

A culture of Bacillus subtilis was used for testing the UV-protectiveeffects of the extracts described herein. Cultures of B. subtilis(0.1-0.2 optical density) were used. 5 mL of these cultures were mixedin Petri dishes with 1, 2, 5, 10, 15, or 20 mL of extracts from theyeast-zinc device; in general, 10 mL was preferred. The B.subtilis/biological device mixtures were exposed to UV radiation (either254 nm or 365 nm, though 254 nm was preferred) while being protectedfrom non-controlled, outside sources of light. UV exposure was carriedout for up to 24 hours at room temperature (24-27° C.). Samples wereremoved at 0, 30, and 60 minutes as well as at 24 hours. Each sampleconsisted of a 200 μL aliquot of the treated mixture, which was platedin nutrient agar and incubated at 37° C. for 24 hours. Growth ofbacteria was determined by the presence of colonies at each samplingtime. FIG. 3 shows sample B. subtilis cultures prior to UV exposure.FIGS. 4A-4C show an untreated control (left petri dish), a lowconcentration of bacteria treated with the extracts described herein andexposed to UV (middle petri dish), and a high concentration of bacteriatreated with the extracts described herein and exposed to UV (rightpetri dish) at different time points (30 min, 1 hour, 24 hours). Treatedbacterial cultures (middle and right dishes) show higher bacterialcolony counts than untreated cultures (left dish), thus demonstratingthe protective effects of the extracts.

Results of additional trials and experiments are shown in FIGS. 5A-5Dand 6A-6B. In these experiments, the effectiveness of a nanopowder formof zinc oxide (particle size<100 nm) at shielding bacterial samples fromUV radiation was compared to extracts from the devices disclosed herein.For these experiments, a 254 nm shortwave UV lamp (Cole Parmer) wasplaced at 20 cm from the samples to be irradiated. Prior to UV exposure,B. subtilis showed complete coverage of the experimental plate, with aconcentration of greater than 1×10⁶ cells (FIG. 5A). After 30 minutes ofexposure to UV light, abnormally shaped colonies were observed for anuntreated control, while complete coverage of the plate was again seenin samples treated with the extracts disclosed herein (FIG. 5B). After 1hour of exposure to UV light, complete cell death was observed inuntreated colonies, whereas complete coverage of the plate was againseen in treated samples (FIG. 5C). After 24 hours of exposure to UVlight, some decline in cell count was seen in treated samples (i.e.,approximately 1×10⁵ cells were present) (FIG. 5D).

A comparative experiment was conducted using zinc oxide nanopowder witha particle size of <100 nm (Sigma-Aldrich). Prior to UV exposure, cellcount in the experimental plate for the sample was approximately 1×10⁶(FIG. 6A). Following 30 minutes of exposure, abnormal colonies wereobserved with a cell count of about 1×10³; after 1 hour of exposure,complete cell death was observed (FIG. 6B).

Example 8: Use of the Yeast-Zinc Device for UV Protection of BacterialCultures

A culture of Bacillus subtilis (ATCC 82) was grown at 30° C. for one totwo days. Aliquots were taken from this culture and subjected todifferent bacteria dilutions and the concentration was determinedspectrophotometrically at the respective optical density (OD) between1.0 to 1.5.

Solutions were made from the above dilutions and mixed with differentconcentrations of extract from Zinc devices, those obtaining differentvolume ratios (i.e. 8:2, 10:2, zinc extract:bacteria). These solutionswere placed in Petri dishes with a total volume of 10 mL and 12 mLrespectably. These solutions were made in triplicates.

The solutions were placed in the UV incubator at 30° C. and samples wereexposed to UV radiation at different times (i.e. 30 minutes, 1 hour, 12hours). In a different experiment, the zinc extract (8 or 10 mL) wasexposed to radiation (e.g., 30 minutes). At each time interval, aliquotsof 1 mL bacterial samples were taken from each replicate (total samplingvolume 3 mL) and fully mixed. Then, 500 μL were taken and placed onNutrient agar by using standard streaking method. Three agar platereplicates were used at each time. The agar plates were incubated at 30°C. for 1-4 days. Bacterial colonies of Bacillus subtilis were viewed andcounted at each time.

Bacterial colonies samples were stained by using standard Gram stainingtechnique and observed in the compound microscope in order to see themorphology of the B. subtilis bacteria.

Table 9 show B. subtilis culture treated with zinc extract and exposedto UV-B radiation (302 nm) showed higher growth by covering the agarplate completely as compared to B. subtilis without exposure to the zincextract, which showed very low growth or no growth at all after exposureto radiation. This confirms the ability of the zinc extract to impart UVprotection.

TABLE 9 Protective effect of zinc extract (500 μg/L) on Bacillussubtilis (ATCC 82) against UV-B radiation (302 nm) at different exposuretimes and extract/bacteria volume ratios Protective effect of ZincDevice on Bacillus subtilis (ATCC 82), Against UV-B at different timesinitial B. subtilis culture alone + no UV-B Colonies covers the platecompletly B. subtilis + Zn Device + B. subtilis alone + UV-B UV-B(10:2,Device extract Bacteria) Count before UV-B expousure Count before UV-Bexpousure Colonies covers the plate completly Colonies covers the platecompletly B. B. B. B. B. B. subtilis + subtilis + subtilis + subtilis +subtilis + subtilis + Zn Zn Zn Zn Zn Zn Device + Device + Device +Device + Device + Device + UV-B UV-B UV-B UV-B UV-B UV-B B. (10:2, (8:2,B. (10:2, (8:2, B. (10:2, (8:2, B. subtilis Device Device subtilisDevice Device subtilis Device Device subtilis alone + extract: extract:alone + extract: extract: alone + extract: extract: alone + UV-BBacteria) Bacteria) UV-B Bacteria) Bacteria) UV-B Bacteria) Bacteria)UV-B Count Count Count 30 30 30 1 1 1 4 before before before minutesminutes minutes hour hour hour hour UV-B UV-B UV-B UV-B UV-B UV-B UV-BUV-B UV-B UV-B expou- expou- expou- expou- expou- expou- expou- expou-expou- expou- sure sure sure sure sure sure sure sure sure sure CPC CPCCPC CPC CPC CPC 4 CPC CPC 0 B. subtilis + Zn Device + UV-B(8:2, Deviceextract Bacteria) Count before UV-B expousure Colonies covers the platecompletly B. subtilis + B. subtilis + B. subtilis + B. subtilis + ZnDevice + Zn Device + Zn Device + Zn Device + UV-B(10:2, UV-B(8:2,UV-B(10:2, UV-B(8:2, Device Device B. subtilis Device Device extract:extract: alone + extract: extract: Bacteria) Bacteria) UV-B Bacteria)Bacteria) 4 hour 4 hour 8 hour 8 hour 8 hour UV-B UV-B UV-B UV-B UV-Bexpousure expousure expousure expousure expousure 217 68 0 1 50 * CPCmeans colonies covers the plate completly

Tables 10 and 11 show B. subtilis culture treated with zinc extract andexposed to UV-B radiation (302 nm) showed higher growth by covering theagar plate completely as compared to B. subtilis without exposure tozinc extract and exposed to UV-B radiation, which showed very low growthor no growth at all after exposure to radiation. This confirms theability of the zinc extract to impart UV protection.

TABLE 10 Protective effect of zinc extract (500 μg/L) on Bacillussubtilis (ATCC 82) against UV-B radiation (302 nm) at different timesand extract/bacteria volume ratios with reapplication at 30 minutesafter radiation exposure Protective effect of Zinc Device on Bacillussubtilis (ATCC 82), Against UV-Bat different times initial B. subtilisculture alone + no UV-B Colonies covers the plate completly B.subtilis + Zn Device + UV-B B. subtilis + Zn Device + UV-B B. subtilisalone + UV-B (10:2, Device extract:Bacteria) (8:2, Device extract:Bacteria) 30 minutes UV-B expousure before R 30 minutes UV-B expousurebefore R 30 minutes UV-B expousure before R 3480,00 Colonies covers theplate completly Colonies covers the plate completly reapplication* after30 minutes UV-B expousure B. B. B. B. B. B. B. B. subtilis + subtilis +subtilis + subtilis + subtilis + subtilis + subtilis + subtilis + Zn ZnZn Zn Zn Zn Zn Zn Device + Device + Device + Device + Device + Device +Device + Device + UV-B UV-B UV-B UV-B UV-B UV-B UV-B UV-B (10:2, (8:2,(10:2, (8:2, (10:2, (8:2, (10:2, (8:2, B. subtilis Device Device B.subtilis Device Device B. subtilis Device Device B. subtilis DeviceDevice alone + extract: extract: alone + extract: extract: alone +extract: extract: alone + extract: extract: UV-B Bacteria) Bacteria)UV-B Bacteria) Bacteria) UV-B Bacteria) Bacteria) UV-B Bacteria)Bacteria) Count Count Count 30 minutes 30 minutes 30 minutes 1 hour 1hour 1 hour 12 hour 12 hour 12 hour before before before UV-B UV-B UV-BUV-B UV-B UV-B UV-B UV-B UV-B reappli- reappli- reappli- expousureexpousure expousure expousure expousure expousure expousure expousureexpousure cation cation cation after R after R after R after R after Rafter R after R after R after R 3480 CPC CPC 80 CPC CPC 0 CPC CPC 0 3312310 *CPC means colonies covers the plate completly

TABLE 11 Protective effect of zinc extract (500 μg/L) on Bacillussubtilis (ATCC 82) against UV-B radiation (302 nm) at different timesand extract/bacteria volume ratios with reapplication at one hour afterradiation exposure Protective effect of Zinc Device on Bacillus subtilis(ATCC 82), Against UV-B at different times initial B. subtilis culturealone + no UV-B Colonies covers the plate completly B.subtilis + ZnDevice + B. subtilis + Zn Device + UV-B (10:2, Device extract: UV-B(8:2, Device extract: B. subtilis alone + UV-B 30 minutes UV-B expousurebefore R 30 minutes UV-B expousure before R 30 minutes UV-B expousurebefore R Colonies covers the plate completly Colonies covers the platecompletly Colonies covers the plate completly B. subtilis + Zn Device +B. subtilis + Zn Device + B.subtilis alone + UV-B UV-B (10:2, Deviceextract: UV-B (8:2, Device extract: 1 hour UV-B expousure before R 1hour UV-B expousure before R 1 hour UV-B expousure before R 350,00Colonies covers the plate completly Colonies covers the plate completlyreapplication* after 1 hour UV-B expousure B. B. B. B. B. B. B. B.subtilis + subtilis + subtilis + subtilis + subtilis + subtilis +subtilis + subtilis + Zn Zn Zn Zn Zn Zn Zn Zn Device + Device + Device +Device + Device + Device + Device + Device + UV-B UV-B UV-B UV-B UV-BUV-B UV-B UV-B (10:2, (8:2, (10:2, (8:2, (10:2, (8:2, (10:2, (8:2, B.subtilis Device Device B. subtilis Device Device B. subtilis DeviceDevice B. subtilis Device Device alone + extract: extract: alone +extract: extract: alone + extract: extract: alone + extract: extract:UV-B Bacteria) Bacteria) UV-B Bacteria) Bacteria) UV-B Bacteria)Bacteria) UV-B Bacteria) Bacteria) Count Count Count 30 minutes 30minutes 30 minutes 1 hour 1 hour 1 hour 4 hour 4 hour 4 hour beforebefore before UV-B UV-B UV-B UV-B UV-B UV-B UV-B UV-B UV-B reappli-reappli- reappli- expousure expousure expousure expousure expousureexpousure expousure expousure expousure cation cation cation after Rafter R after R after R after R after R after R after R after R 350 CPCCPC 28 CPC CPC 14 CPC CPC 0 CPC CPC *CPC means colonies covers the platecompletly

Table 12 shows B. subtilis culture treated with the zinc extract andexposed to UV-A radiation (365 nm) showed higher growth by covering theagar plate completely as compared to B. subtilis without exposure tozinc extract, which showed very low growth or no growth at all afterexposure to UV-A radiation. This confirms the ability of the zincextract to impart UV protection.

TABLE 12 Protective effect of zinc extract (~500 μg/L ) on Bacillussubtilis (ATCC 82) against UV-A radiation (365 nm) at different timesand extract/bacteria volume ratios Protective effect of Zinc Device onBacillus subtilis (ATCC 82), Against UV-A at different times initial B.subtilis culture alone- no UV-A Colonies covers the plate completly B.subtilis alone + UV-A B. subtilis + Zn Device + UV-A (4:1, Deviceextract: Bacteria) Count before UV-A expousure Count before UV-Aexpousure Colonies covers the plate completly Colonies covers the platecompletly B. B. B. B. B. B. B. subtilis + subtilis + subtilis +subtilis + subtilis + subtilis + subtilis + Zn Zn Zn Zn Zn Zn ZnDevice + Device + Device + Device + Device + Device + Device + B. UV-A(4:1, B. UV-A B. UV-A UV-A UV-A UV-A UV-A subtilis Device subtilis (4:1,subtilis (4:1, B. (4:1, B. (4:1, B. (4:1, B. (4:1, alone + extract:alone + Device alone + Device subtilis Device subtilis Device subtilisDevice subtilis Device UV-A Bacteria) UV-A extract: UV-A extract:alone + extract: alone + extract: alone + extract: alone + extract:Count Count 30 Bacteria) 45 Bacteria) UV-A Bacteria) UV-A Bacteria) UV-ABacteria) UV-A Bacteria) before before minutes 30 minutes minutes 45minutes 1 hour 1 hour 4 hour 4 hour 8 hour 8 hour 24 hour 24 hour UV-AUV-A UV-A UV-A UV-A UV-A UV-A UV-A UV-A UV-A UV-A UV-A UV-A UV-A expou-expou- expou- expou- expou- expou- expou- expou- expou- expou- expou-expou- expou- expou- sure sure sure sure sure sure sure sure sure suresure sure sure sure CPC CPC 60 CPC 29 CPC 25 210 0 CPC 86 CPC 104 CPC *CPC means colonies covers the plate completly

Table 13 shows B. subtilis culture treated with zinc extract and exposedto UV-C radiation (254 nm) showed higher growth by covering the agarplate completely as compared to B. subtilis without exposure to zincextract, which showed very low growth or no growth at all after exposureto UV-C. This confirms the ability of the zinc extract to impart UVprotection.

TABLE 13 Protective effect of zinc extract (1.3 mg/L) on Bacillussubtilis (ATCC 82) against UV-C radiation (254 nm) at different timesand extract/bacteria volume ratios Protective effect of Zinc Device onBacillus subtilis (ATCC 82), Against UV-C at different times initial B.subtilis culture alone + no UV-C Colonies covers the plate completly B.subtilis + Zn Device + B. subtilis alone + UV-C UV-C (4:1, Deviceextract: Count before UV-C expousure Count before UV-C expousureColonies covers the plate completly Colonies covers the plate completlyB. subtilis + B. subtilis + B. subtilis + B. subtilis + Zn Device + ZnDevice + Zn Device + Zn Device + UV-C (4:1, UV-C UV-C UV-C B. subtilisDevice (4:1, (4:1, (4:1, alone + extract: B. subtilis Device B. subtilisDevice B. subtilis Device UV-C Bacteria) alone + extract: alone +extract: alone + extract: Count Count UV-C Bacteria) UV-C Bacteria) UV-CBacteria) before before 30 minutes 30 minutes 1 hour 1 hour 24 hour 24hour UV-C UV-C UV-C UV-C UV-C UV-C UV-C UV-C expousure expousureexpousure expousure expousure expousure expousure expousure CPC CPC 46CPC 0 CPC 0 368 * CPC means colonies covers the plate completly

Example 9: Protective Effect of Oxidized Zinc on Fibroblast CellProduction of Oxidized Zinc

The following steps were performed to produce and isolate the oxidizedzinc extract:

1. Fermentation in yeast malt medium with 2% of raffinose and inductionwith 1% of galactose at 30° C. for 72 hours.2. Sonication: 7 times for 2.5 minutes.3. Filtration of supernatant by 8 μm, 3 μm, 2 μm and 1.2 μm.

Procedure

Skin fibroblast cells (ATCC 2522-CRL) were used as a model for humanskin and were maintained in culture media for propagation and renewalfollowing ATCC recommendations. The propagation medium is based onATCC-formulated Eagles's Minimum Essential Medium, Catalog No 30-2003.Fetal bovine serum was added to the medium to a final concentration of10%. The medium was also renewed according to ATCC instructions. Thismedium is made of 0.025% trypsin, 0.03% EDTA solution. Cultures offibroblast cells (ATCC 2522-CRL) were grown at 37° C. and 5% CO₂.

Oxidized zinc extract was applied to fibroblast culture with differentconcentrations (300-500 μg/L) of oxidized zinc extract at differentratios extract/skin cells (1:1, 2:1, 3:1, 5:4) (Zn device extract:skincells) with 5:4 as the preferred ratio. This mixture was then exposed toUV-B radiation (302 nm) for different times and incubated at 37° C. and5% of CO₂. Each experiment was performed in triplicate.

Aliquots of fibroblast cells were harvested and subjected to microscopicanalysis. Standard procedures were used to count dead, live, andapoptotic cells by staining the cells with trypan blue (1:1,trypan:sample) and viewing them under a compound microscope. Cells werecounted at 20× magnification using several (16 each time) microscopicfield views. Results are presented as the average of these 16microscopic optical field samples.

Results

Results are presented in Tables 14 and 15 as the average of these 16microscopic optical field samples. Alive cells are elongated withoutblue pigmentation. Dead cells are spherical with intense bluepigmentation. Apoptotic cells are slightly curved with slight or nolight blue pigmentation. Initial Culture of Fibroblast Cells: alivecells: 87; death cells: 3; apoptotic cells: 3

When sunlight passes through the atmosphere, the ozone, water vapor,oxygen and carbon dioxide absorb approximately 90% of UVB radiation,hence reducing the amount of exposure to UVB radiation; however, thisUVB wavelength also causes damage to human skin. (World HealthOrganization/WHO/SDE/OEH/02.2, 2003). As shown in the tables below, theoxidized zinc is highly effective in protecting the skin against UVBradiation, considering that the anti-UVB experiments were performedunder continued direct exposure to UVB radiation for the entire time ofthe experiment.

TABLE 14 Number of alive, dead and apoptotic fibroblast cells atdifferent times. Protective effect of Zinc Device on SC, Against UV-B atdifferent times SCC + Water SCC + Zn Device (Control) (Treatment) Typeof cell 45 minutes 2 hour 4 hour 45 minutes 2 hour 4 hour Alive Cells 403 0 51 22 14 (average) Dead Cells 3 18 13 3 5 4 (average) Apoptotic 3 00 5 8 6 Cells (average)

TABLE 15 Percentage of alive, dead and apoptotic fibroblast cells atdifferent times. Protective effect of Zinc Device on SC, Against UV-B atdifferent times SCC + Water SCC + Zn Device (Control) (Treatment) Typeof cell 45 minutes 2 hour 4 hour 45 minutes 2 hour 4 hour Alive Cells 8614 0 86 63 58 (Percentage) Dead Cells 7 86 100 5 14 17 (Percentage)Apopoptotic 7 0 0 8 23 25 Cells (Percentage)

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the compounds, compositions, and methods described herein.

Various modifications and variations can be made to the compounds,compositions, and methods described herein. Other aspects of thecompounds, compositions, and methods described herein will be apparentfrom consideration of the specification and practice of the compounds,compositions, and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

1. A DNA construct comprising the following genetic components: a. agene that expresses zinc-related protein; b. a gene that expressesalkaline phosphatase; and c. a gene that expresses alcoholdehydrogenase.
 2. The DNA construct of claim 1, wherein the gene thatexpresses zinc-related protein has SEQ ID NO. 1 or at least 70% homologythereto.
 3. The DNA construct of claim 1, wherein the gene thatexpresses alkaline phosphatase has SEQ ID NO. 2 or at least 70% homologythereto.
 4. The DNA construct of claim 1, wherein the gene thatexpresses alcohol dehydrogenase has SEQ ID NO. 3 or at least 70%homology thereto.
 5. The DNA construct of claim 1, wherein the DNAconstruct further comprises a gene that expresses lipase.
 6. The DNAconstruct of claim 5, wherein the gene that expresses lipase has SEQ IDNO. 6 or at least 70% homology thereto.
 7. (canceled)
 8. (canceled) 9.The DNA construct of claim 7, wherein a GAL1 promoter is positionedbefore the gene that expresses zinc-related protein, the gene thatexpresses alkaline phosphatase, and the gene that expresses alcoholdehydrogenase. 10-18. (canceled)
 19. The DNA construct of claim 1,wherein the DNA construct comprises from 5′ to 3′ the following geneticcomponents in the following order: (a) a gene that expresses azinc-related protein, (b) a gene that expresses an alkaline phosphatase,and (c) a gene that expresses an alcohol dehydrogenase.
 20. The DNAconstruct of claim 1, wherein the DNA construct comprises from 5′ to 3′the following genetic components in the following order: (a) a gene thatexpresses a zinc-related protein, (b) a gene that expresses a lipase,(c) a gene that expresses an alkaline phosphatase, and (d) a gene thatexpresses an alcohol dehydrogenase.
 21. The DNA construct of claim 1,wherein the DNA construct comprises from 5′ to 3′ the following geneticcomponents in the following order: (a) a GAL1 promoter, (b) a gene thatexpresses a zinc-related protein, (c) a CYC1 terminator, (d) a GAL1promoter, (e) a gene that expresses an alkaline phosphatase, (f) a CYC1terminator, (g) a GAL1 promoter, (h) a gene that expresses an alcoholdehydrogenase, (i) a CYC1 terminator.
 22. The DNA construct of claim 1,wherein the DNA construct comprises from 5′ to 3′ the following geneticcomponents in the following order: (a) a gene that expresses azinc-related protein, (b) a CYC1 terminator, (c) a GAL1 promoter, (d) agene that expresses a lipase, (e) a CYC1 terminator, (f) a GAL1promoter, (g) a gene that expresses an alkaline phosphatase, (h) a CYC1terminator, (i) a GAL1 promoter, (j) a gene that expresses an alcoholdehydrogenase, and (k) a CYC1 terminator.
 23. The DNA construct of claim1, wherein the DNA construct comprises from 5′ to 3′ the followinggenetic components in the following order: (a) a GAL1 promoter, (b) agene that expresses a zinc-related protein having SEQ ID NO. 1 or atleast 70% homology thereto, (c) a CYC1 terminator, (d) a GAL1 promoter,(e) a gene that expresses an alkaline phosphatase having SEQ ID NO. 2 orat least 70% homology thereto, (f) a CYC1 terminator, (g) a GAL1promoter, (h) a gene that expresses an alcohol dehydrogenase having SEQID NO. 3 or at least 70% homology thereto, and (i) a CYC1 terminator.24. The DNA construct of claim 1, wherein the DNA construct comprisesfrom 5′ to 3′ the following genetic components in the following order:(a) a gene that expresses a zinc-related protein having SEQ ID NO. 1 orat least 70% homology thereto, (b) a CYC1 terminator, (c) a GAL1promoter, (d) a gene that expresses a lipase having SEQ ID NO. 6, (e) aCYC1 terminator, (f) a GAL1 promoter, (g) a gene that expresses analkaline phosphatase having SEQ ID NO. 2 or at least 70% homologythereto, (h) a CYC1 terminator, (i) a GAL1 promoter, (j) a gene thatexpresses an alcohol dehydrogenase having SEQ ID NO. 3 or at least 70%homology thereto, and (k) a CYC1 terminator.
 25. The DNA construct ofclaim 1, wherein the DNA construct has SEQ ID NO. 5 or at least 70%homology thereto.
 26. The DNA construct of claim 5, wherein the DNAconstruct has SEQ ID NO. 7 or at least 70% homology thereto.
 27. Avector comprising the DNA construct of claim
 1. 28. The vector of claim27, wherein the vector is a plasmid.
 29. The vector of claim 28, whereinthe plasmid is pWLneo, pSV2cat, pOG44, pXT1, pSG, pSVK3, pBSK, pBSKII,pYES, pYES2, pUC, or pUC19.
 30. (canceled)
 31. A biological devicecomprising host cells transformed with the DNA construct of claim
 1. 32.The biological device of claim 31, wherein the host cells comprise fungior bacteria.
 33. (canceled)
 34. (canceled)
 35. (canceled)
 36. A methodfor producing a composition comprising oxidized zinc, the methodcomprising growing the biological device of claim 1 for a sufficienttime to produce the composition.
 37. The method of claim 36, the methodfurther comprising lysing the host cells to produce a lysed composition,and (2) separating the oxidized zinc from the lysed composition.
 38. Acomposition produced by the method of claim
 36. 39-44. (canceled)
 45. Apaint, stain, dye, or coating comprising the composition of claim 38.46. A cosmetic, nutritional, or pharmaceutical preparation comprisingthe composition of claim
 38. 47. The cosmetic, nutritional, orpharmaceutical composition of claim 46, wherein the composition is anastringent, a sun cream, a dental cement, a nutritional supplement, or acold-prevention lozenge. 48-51. (canceled)
 52. An agricultural productor plant coated with the extract of claim
 38. 53. The agriculturalproduct of claim 52, wherein the agricultural product comprises fruits,leaves, seeds, flowers, grains, nuts, stems, vegetables, or mushrooms.54. An article coated with the extract of claim 38, the extract isdispersed throughout the article, or a combination thereof.
 55. Thearticle of claim 54, wherein the article is made of glass, fiberglass,plastic, metal, wood, fabric, foam, rubber, latex, silicone, or anycombination thereof.
 56. A method of reducing or preventing exposure ofan item to UV radiation comprising applying to the item the extract ofclaim
 38. 57. The method of claim 56, wherein the extract blocks atleast 50% of UV radiation from contacting the item.
 58. The method ofclaim 56, wherein the extract blocks at least approximately 50% oflongwave UV radiation from contacting the item.
 59. The method of claim56, wherein the extract blocks at least approximately 50% of shortwaveUV radiation from contacting the item.
 60. The method of claim 56,wherein the item comprises the skin of a subject.
 61. The method ofclaim 56, wherein the item comprises an agricultural product.
 62. Themethod of claim 56, wherein the item comprises a construction material,and aeronautical, or an aerospace material.
 63. A method for increasingthe level of oxidized zinc in a subject comprising administering to thesubject the extract of claim
 38. 64. The method of claim 63, whereinsubject has cancer.
 65. The method of claim 63, wherein subject isundergoing chemotherapy.
 66. The method of claim 63, wherein the extractis administered orally or parenterally.
 67. A health supplementcomprising the extract of claim 38.